Irreversible cysteine protease inhibitors containing vinyl groups conjugated to electron withdrawing groups

ABSTRACT

Irreversible cysteine protease inhibitors based upon an alkene bond being conjugated to an electron withdrawing group are disclosed. The inhibitor structure also provides a targeting peptide which is specific for different cysteine proteases. The method of making the inhibitors, and methods of using the inhibitors to inhibit cysteine proteases and for therapy are disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.08/700,518, filed Aug. 23, 1996, which issued as U.S. Pat. No.5,976,858, Nov. 2, 1999, which is a national phase entry ofPCT/US95/02252, filed Feb. 24, 1995, which is a continuation of U.S.application Ser. No. 08/202,051 filed Feb. 25, 1994, abandoned.

FIELD OF THE INVENTION

The invention relates to novel cysteine protease inhibitors. Theinhibitors are specific to cysteine proteases and do not inhibit serine,aspartyl or zinc protease.

BACKGROUND OF THE INVENTION

Cysteine or thiol proteases contain a cysteine residue at the activesite responsible for proteolysis. Since cysteine proteases have beenimplicated in a number of diseases, including arthritis, musculardystrophy, inflammation, tumor invasion, glomerulonephritis, malaria,and other parasite-borne infections, methods for selectively andirreversibly inactivating them provide opportunities for new drugcandidates. See, for example, Meijers, M. H. M. et al., Agents Actions(1993), 39 (Special Conference Issue), C219; Machleidt, W. et al,Fibrinolysis (1992), 6 Suppl. 4, 125; Sloane, B. F. et al., Biomed.Biochim. Acta (1991), 50, 549; Duffy, M. J., Clin. Exp. Metastasis(1992), 10, 145; Rosenthal, P. J., Wollish, W. S., Palmer, J. T.,Rasnick, D., J. Clin. Investigations (1991), 88, 1467; Baricos, W. H. etal, Arch. Biochem. Biophys. (1991), 288, 468; Thornberry, N. A. et al.,Nature (1992), 356, 768.

Low molecular weight inhibitors of cysteine proteases have beendescribed by Rich, Proteinase Inhibitors (Chapter 4, “Inhibitors ofCysteine Proteinases”), Elsevier Science Publishers (1986). Suchinhibitors include peptide aldehydes, which form hemithioacetals withthe cysteine of the protease active site. The disadvantage of aldehydesis their in vivo and chemical instabilities.

Aldehydes have been transformed into α,β-unsaturated esters and sulfonesby means of the Wadsworth-Emmons-Horner modification of the Wittigreaction (Wadsworth, W. S. and Emmons, W. D. (J. Am. Chem. Soc. (1961),83, 1733: Equation 1).

where

R=alkyl, aryl, etc.

EWG=COOEt, SO₂Me, etc.

α,β-unsaturated esters (Hanzlik et al., J. Med. Chem., 27(6):711-712(1984), Thompson et al., J. Med. Chem. 29:104-111 (1986), Liu et al., J.Med. Chem., 35(6):1067 (1992)) and an α,β-unsaturated sulfones (Thompsonet al., supra, Liu et al., supra) were made using this method and testedas inhibitors of two cysteine proteases, papain and dipeptidylamino-peptidase I (also called cathepsin C). However, the inhibition ofpapain by these α,β-unsaturated compounds showed poor inhibition,evidenced by second order rate constants from less than 1 M⁻¹sec⁻¹ toless than 70 M⁻¹sec⁻¹ for the α,β-unsaturated esters, and from less than20 M⁻¹sec⁻¹ to less than 60 M⁻¹sec⁻¹ for the sulfone.

In addition, this chemistry has not been demonstrated with derivativesof α-amino acids other than those corresponding to glycine, or in thecase of the ester, phenylalanine. Thus the chirality of these compoundsis non-existent for the glycine derivatives and unclear for thephenylalanine derivatives. This is significant since inhibition of anenzyme generally requires a chiral compound.

Additional methods for selectively and irreversibly inhibiting cysteineproteases have relied upon alkylation by peptide α-fluoromethyl ketones(Rasnick, D., Anal. Biochem. (1985), 149, 416), diazomethylketones(Kirschke, H., Shaw, E. Biochem. Biphys. Res. Commun. (1981), 101, 454),acyloxymethyl ketones (Krantz, A. et al., Biochemistry, (1991), 30,4678; Krantz, A. et al., U.S. Pat. No. 5,055,451, issued Oct. 8, 1991),and ketosulfonium salts (Walker, B., Shaw, E., Fed. Proc. Fed. Am. Soc.Exp. Biol., (1985), 44, 1433). The proposed mechanism of inactivationrelies upon irreversible inactivation of the active site thiol group viaalkylation, as depicted in Equation 2.

where

PG=protecting group

R₁, R₂=amino acid side chains

X=Cl, F, N₂, OC(O)R, (+)S(CH₃)₃

Other families of cysteine protease inhibitors include epoxysuccinylpeptides, including E-64 and its analogs (Hanada, K. et al., Agric.Biol. Chem (1978), 42, 523; Sumiya, S. et al., Chem. Pharm. Bull.((1992), 40, 299 Gour-Salin, B. J. et al., J. Med. Chem., (1993), 36,720), α-dicarbonyl compounds, reviewed by Mehdi, S., BioorganicChemistry, (1993), 21, 249, and N-peptidyl-O-acyl hydroxamates (Bromme,D., Neumann, U., Kirschke, H., Demuth, H-U., Biochim. Biophys. Acta,(1993), 1202, 271.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide novel cysteineprotease inhibitors that function irreversibly, resulting in largesecond order rate constants for the overall inhibition reaction.Accordingly, it is an object to provide these novel cysteine proteaseinhibitors that can be used to inhibit cysteine proteases selectively,and are thus useful in a variety of therapeutic applications.

In accordance with the foregoing objects, the present invention providescysteine protease inhibitors comprising a targeting group linked to analkene bond electronically conjugated with an electron withdrawing group(EWG). The second order rate constant for inhibition of a cysteineprotease with the inhibitor, expressed as k_(irr)/K_(I), preferably isat least about 1000 M⁻¹sec⁻¹.

An additional aspect of the present invention is to provide chiralcysteine protease inhibitors comprising a targeting group linked to analkene bond conjugated with an EWG. Additionally provided are thesechiral cysteine protease inhibitors wherein the second order rateconstant for inhibition of a cysteine protease with the inhibitor,expressed as k_(irr)/K_(I), is at least about 1000 M⁻¹sec⁻¹.

In a further aspect of the present invention, a cysteine proteaseinhibitor is provided with the formula:

In this formula, R₁₀ is hydrogen, a peptide amino end blocking group, apeptide residue with or without an amino end blocking group, a singleamino acid residue with or without an amino end blocking group or alabel. X and R₁₁ are amino side chains, with either (R) or (S)configuration. The A—B linkage is a peptide or peptidomimetic linkage,and EWG is an electron withdrawing group. Also provided are cysteineprotease inhibitors wherein the second order rate constant forinhibition of a cysteine protease with the inhibitor, expressed ask_(irr)/K_(I), is at least about 1000 M⁻¹sec⁻¹.

Also provided are labelled cysteine protease inhibitors, and cysteineprotease inhibitors that contain additional targeting groups linked tothe EWG.

In a further aspect of the present invention, cysteine proteaseinhibitors are provided wherein the EWG is a moiety or group that willwork in the Wadsworth-Emmons reaction for olefin synthesis when directlyattached to a methylenephosphonate species. Thus the EWG group, whenfunctionally included as a component of the cysteine protease inhibitor,may be selected from the group consisting of vinylogous esters,vinylogous sulfones, vinylogous carboxylates, vinylogous amides,vinylogous phosphonates, vinylogous ketones, vinylogous nitriles,vinylogous sulfoxides, vinylogous sulfonamides, vinylogous sulfinamides,vinylogous sulfonates and vinylogous sulfoximines.

An additional aspect of the present invention relates to methods formaking a cysteine protease inhibitor. The method comprises: a) aprotected α-amino aldehyde is coupled with a Wadsworth-Emmons reagentcontaining an EWG to form a cysteine protease inhibitor intermediate; b)the cysteine protease inhibitor intermediate is the n deprotected at theN-terminus; and c) the deprotected cysteine protease inhibitorintermediate is then coupled to N-terminally protected amino acids.

The invention also includes a method for inhibiting a cysteine protease,comprising irreversibly binding an cysteine protease inhibitor to theprotease.

The invention further provides a method of treating cysteineproteane-associated disorders, comprising administering atherapeutically effective dose of a cysteine protease inhibitor to apatient. Thus, pharmaceutical compositions of cysteine proteaseinhibitors are also provided.

Additionally, the invention provides methods of detecting a cysteineprotease in a sample, comprising assaying the sample in the presence andabsence of a cysteine protease inhibitor of the present invention andcalculating the difference in activity due to the protease.

Further provided are cysteine protease inhibitors with the formula:

In this formula, R₁₀ is hydrogen, a peptide amino end blocking group, apeptide residue with or without an amino end blocking group, a singleamino acid residue with or without an amino end blocking group, or alabel. X and R₁₁ are amino side chains, with either (R) or (S)configuration. The A—B linkage is a peptide or peptidomimetic linkage,and EWG is an electron withdrawing group.

An additional aspect of the invention provides cysteine proteaseinhibitors with the formula:

In this formula, R₁₀ is hydrogen, a peptide amino end blocking group, apeptide residue with or without an amino end blocking group, a singleamino acid residue with or without an amino end blocking group or alabel. X and R₁₁ are amino side chains, with either (R) or (S)configuration. The A—B linkage is a peptide or peptidomimetic linkage,and EWG is an electron withdrawing group.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to novel cysteine protease inhibitors. Itis believed that the inhibitors function to inactivate cysteineproteases based on the following mechanism:

It is believed that the enzyme is thus irreversibly inhibited by 1,4conjugate addition of the nucleophilic thiol group. This mechanism isthus distinguished from the alkylation mechanism depicted in Equation 2.

This mechanism permits specificity of the enzyme inhibitors for cysteineproteases over serine, aspartyl, and zinc proteases, by virtue of adifferent mechanism. The mechanisms of serine protease action have beendescribed by Walsh, C., in “Enzymatic Reaction Mechanisms” pp. 94-97,W.H. Freeman and Co., San Francisco, 1979. The serine at the active sitereacts with the carbonyl of the substrate, forming a tetrahedralintermediate. The inhibitors of this invention have no carbonyl at thesite of nucleophilic attack, and are not susceptible to attack by serineproteases.

Cysteine proteases are a family of proteases that bear a thiol group atthe active site. These proteases are found in bacteria, viruses,eukaryotic microorganisms, plants, and animals. Cysteine proteases maybe generally classified as belonging to one of four or more distinctsuperfamilies. Examples of cysteine proteases that may be inhibited bythe novel cysteine protease inhibitors of the present invention include,but are not limited to, the plant cysteine proteases such as papain,ficin, aleurain, oryzain and actinidain; mammalian cysteine proteasessuch as cathepsins B, H, J, L, N, S, T and C, (cathepsin C is also knownas dipeptidyl peptidase I), interleukin converting enzyme (ICE),calcium-activated neutral proteases, calpain I and II; viral cysteineproteases such as picornian 2A and 3C, aphthovirus endopeptidase,cardiovirus endopeptidase, comovirus endopeptidase, potyvirusendopeptidases I and II, adenovirus endopeptidase, the twoendopeptidases from chestnut blight virus, togavirus cysteineendopeptidase, as well as cysteine proteases of the polio andrhinoviruses; and cysteine proteases known to be essential for parasitelifecycles, such as the proteases from species of Plasmodia, Entamoeba,Onchocera, Trypansoma, Leishmania, Haemonchus, Dictyostelium,Therileria, and Schistosoma, such as those associated with malaria (P.falciparium), trypanosomes (T. cruzi, the enzyme is,also known ascruzain or cruzipain), murine P. vinckei, and the C. elegans cysteineprotease. For an extensive listing of cysteine proteases that may beinhibited by the cysteine protease inhibitors of the present invention,see Rawlings et al., Biochem. J. 290:205-218 (1993), hereby expresslyincorporated by reference.

Accordingly, inhibitors of cysteine proteases are useful in a widevariety of applications. For example, the inhibitors of the presentinvention are used to quantify the amount of cysteine protease presentin a sample, and thus are used in assays and diagnostic kits for thequantification of cysteine proteases in blood, lymph, saliva, or othertissue samples, in addition to bacterial, fungal, plant, yeast, viral ormammalian cell cultures. Thus in a preferred embodiment, the sample isassayed using a standard protease substrate. A cysteine proteaseinhibitor is added, and allowed to bind to any cysteine proteasepresent. The protease assay is then rerun, and the loss of activity iscorrelated to cysteine protease activity using techniques well known tothose skilled in the art.

The cysteine protease inhibitors are also useful to remove or inhibitcontaminating cysteine proteases in a sample. For example, the cysteineprotease inhibitors of the present invention are added to samples whereproteolytic degradation by contaminating cysteine proteases isundesirable. Alternatively, the cysteine protease inhibitors of thepresent invention may be bound to a chromatographic support, usingtechniques well known in the art, to form an affinity chromatographycolumn. A sample containing an undesirable cysteine protease is runthrough the column to remove the protease.

In a preferred embodiment, the cysteine protease inhibitors are usefulfor inhibiting cysteine proteases implicated in a number of diseases. Inparticular, cathepsins B, L, and S, cruzain, and interleukin 1βconverting enzyme are inhibited. These enzymes are examples of lysosomalcysteine proteases implicated in a wide spectrum of diseasescharacterized by tissue degradation. Such diseases include, but are notlimited to, arthritis, muscular dystrophy, inflammation, tumor invasion,glomerulonephritis, parasite-borne infections, Alzheimer's disease,periodontal disease, and cancer metastasis. For example, mammalianlysosomal thiol proteases play an important role in intracellulardegradation of proteins and possibly in the activation of some peptidehormones. Enzymes similar to cathepsins B and L are released from tumorsand may be involved in tumor metastasis. Cathepsin L is present indiseased human synovial fluid and transformed tissues. Similarly, therelease of cathepsin B and other lysosomal proteases frompolymorphonuclear granulocytes and macrophages is observed in trauma andinflammation.

The cysteine protease inhibitors also find application in a multitude ofother diseases, including, but not limited to, gingivitis, malaria,leishmaniasis, filariasis, and other bacterial and parasite-borneinfections. The compounds also offer application in viral diseases,based on the approach of inhibiting proteases necessary for viralreplication. For example, many picornoviruses including poliovirus, footand mouth disease virus, and rhinovirus encode for cysteine proteasesthat are essential for cleavage of viral polyproteins.

Additionally, these compounds offer application in disorders involvinginterleukin-1β converting enzyme (ICE), a cysteine protease responsiblefor processing interleukin 1β; for example, in the treatment ofinflammation and immune based disorders of the lung, airways, centralnervous system and surrounding membranes, eyes, ears, joints, bones,connective tissues, cardiovascular system including the pericardium,gastrointestinal and urogenital systems, the skin and the mucosalmembranes. These conditions include infectious diseases where activeinfection exists at any body site, such as meningitis and salpingitis;complications of infections including septic shock, disseminatedintravascular coagulation, and/or adult respiratory distress syndrome;acute or chronic inflammation due to antigen, antibody and/or complementdeposition; inflammatory conditions including arthritis, chalangitis,colitis, encephalitis, endocarditis, glomerulonephritis, hepatitis,myocarditis, pancreatitis, pericarditis, reperfusion injury andvasculitis. Immune-based diseases include but are not limited toconditions involving T-cells and/or macrophages such as acute anddelayed hypersensitivity, graft rejection, and graft-versus-hostdisease; auto-immune diseases including Type I diabetes mellitus andmultiple sclerosis. Bone and cartilage reabsorption as well as diseasesresulting in excessive deposition of extracellular matrix such asinterstitial pulmonary fibrosis, cirrhosis, systemic sclerosis, andkeloid formation may also be treated with the inhibitors of the presentinvention. The inhibitors may also be useful in the treatment of certaintumor that produce IL 1 as an autocrine growth factor and in preventingthe cachexia associated with certain tumors. Apoptosis and cell deathare also associated with ICE and may be treated with the inhibitors ofthe present invention.

Furthermore, the cysteine protease inhibitors of the present inventionfind use in drug potentiation applications. For example, therapeuticagents such as antibiotics or antitumor drugs can be inactivated throughproteolysis by endogeneous cysteine proteases, thus rendering theadministered drug less effective or inactive. For example, it has beenshown that bleomycin, an antitumor drug, can be hydrolyzed by bleomycinhydrolase, a cysteine protease (see Sebti et al., Cancer Res. January1991, pages 227-232). Accordingly, the cysteine protease inhibitors ofthe invention may be administered to a patient in conjunction with atherapeutic agent in order to potentiate or increase the activity of thedrug. This co-administration may be by simultaneous administration, suchas a mixture of the cysteine protease inhibitor and the drug, or byseparate simultaneous or sequential administration.

In addition, cysteine protease inhibitors have been shown to inhibit thegrowth of bacteria, particularly human pathogenic bacteria (see Bjorcket al., Nature 337:385 (1989)). Accordingly, the cysteine proteaseinhibitors of the present invention may be used as antibacterial agentsto retard or inhibit the growth of certain bacteria.

The cysteine protease inhibitors of the invention also find use asagents to reduce the damage of bacterial cysteine proteases to hostorganisms. For example, staphylococcus produces a very activeextracellular cysteine protease which degrades insoluble elastin,possibly contributing to the connective tissue destruction seen inbacterial infections such as septicemia, septic arthritis and otitis.See Potempa et al., J. Biol. Chem. 263(6):2664-2667 (1988). Accordingly,the cysteine protease inhibitors of the invention may be used to treatbacterial infections to prevent tissue damage.

The present invention generally provides new peptide-based andpeptidomimetic cysteine protease inhibitors for use as irreversible,mechanism-based cysteine protease inhibitors. In the preferredembodiment, these cysteine protease inhibitors are Michael acceptors. Bythe term “Michael acceptor” or grammatical equivalents herein is meantan alkene conjugated with an electron-withdrawing group, such as anα,β-unsaturated, vinylogous electron withdrawing group.

The cysteine protease inhibitors are comprised of a targeting grouplinked to an alkene conjugated with an EWG. A preferred embodimentexhibits the structure shown below in Formula 1:

wherein

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid residuewith or without an amino end blocking group or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

A—B is a peptide linkage; and

EWG is an electron withdrawing group.

By the term “targeting group”, or grammatical equivalents, herein ismeant a portion of a cysteine protease inhibitor that allows the bindingof the inhibitor to a cysteine protease. In a preferred embodiment, thetargeting group of a cysteine protease inhibitor comprises at least oneamino acid side chain. In the preferred embodiment, the targeting groupcomprises at least two amino acids linked via a peptide bond. In apreferred embodiment, the carbon to which the R₁₁ amino acid side chainis attached is directly attached to the carbon-carbon double bond. Thetargeting group may include up to about 15 amino acids, althoughcysteine protease inhibitors are generally from 1 to 7 amino acids,since smaller inhibitors are usually desired in therapeuticapplications.

The targeting group may comprise naturally occurring amino acids andpeptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids, although preferably a singlecysteine protease inhibitor does not contain more than one non-naturallyoccurring amino acid side chain. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The term as used herein also refers toportions of an amino acid, such as an amino acid side chain. The sidechains may be in either the (R) or the (S) configuration. In thepreferred embodiment, the amino acids are in the (S) or L-configuration.

If non-naturally occurring side chains are used, non-amino acidsubstituents may be used, for example to prevent or retard in vivodegradations. Such non-amino acid substituents will normally include,but are not limited to, an alkyl, a cycloalkyl, a cycloalkylalkyl, acycloalkylalkenyl, an aryl, an aralkyl, an alkoxy, a heteroaryl, aheteroarylalkyl, or a heteroarylalkenyl. In such an instance, alkyl ispreferably of 1 to 5 carbon atoms, preferably branched, particularlyisobutyl. Cycloalkyl preferably is of 3 to 7 carbon atoms, preferablycyclopentyl or cyclohexyl. Cycloalkylalkyl or cycloalkylalkenylpreferably is of 3 to 7 carbon atoms in the cycloalkyl, particularly 5or 6 carbon atoms, and of 1 to 5 carbon atoms, particularly 1 carbonatom, in the alkyl or alkylene moieties thereof. Aryl preferably isphenyl. Aralkyl preferably is phenylalkyl of 7 to 12 carbon atoms,particularly benzyl and phenylethyl. Alkoxy preferably is of 1 to 5carbon atoms, preferably methoxy. Acyloxy preferably is of 2 to 6 carbonatoms, preferably acetoxy. Heteroaryl preferably is pyridinyl,especially 4-pyridinyl, thienyl, especially 2-thienyl, or furyl,especially 2-furyl. Heteroarylalkyl and heteroalkenyl preferably has 1to 6 carbon atoms, especially 1 carbon atom in the alkyl or alkylenemoieties thereof. The heteroaryl moiety of heteroarylalkyl andheteroarylakylene preferably has the significances indicated above aspreferred for heteroaryl. The optional substituents of an aryl oraralkyl moiety preferably are one or two groups alkyl of 1 to 5 carbonatoms, alkoxy of 1 to 5 carbon atoms, halogen of atomic number of from 9to 35, hydroxy and/or amino, preferably one or two groups methyl,methoxy, chlorine, bromine, fluorine, hydroxy or amino, particularly onehydroxy, amino, chlorine, bromine, or fluorine, optionally in protectedform where appropriate, halogen-substituted alkyl of 1 to 5 carbonatoms, particularly trifluoromethyl.

The specific amino acids comprising the targeting group are initiallydetermined by the rules governing substrate specificity in cysteineproteases. That is, combinations of amino acids that confer specificityto the enzyme to be inhibited will be used.

It is to be understood that the order of the amino acid side chainswithin the inhibitor is significant in conferring inhibitor targeting.Thus, as is well known for substrates, the amino acid side chain of thetargeting group closest to the alkene bond, generally referred to hereinas R₁₁, will occupy the S₁ position of the enzyme's substrate bindingsite when the inhibitor is bound to the enzyme. That is, the R₁₁ aminoacid side chain of the targeting group is the P₁ residue of theinhibitor. Similarly, the amino acid side chain of the targeting groupsecond from the alkene bond, generally referred to herein as “X”, willoccupy the S₂ position of the enzyme's substrate binding site when theinhibitor is bound to the enzyme, and thus is the P₂ residue. Ifpresent, additional amino acid side chains of the inhibitor will occupythe P₃, P₄, etc. positions.

In a preferred embodiment, additional targeting residues orspecificity-enhancing residues are attached to the EWG, to occupy theS₁′, S₂′, etc. position of the enzyme's substrate binding site. Theseadditional targeting groups are considered the P₁′, P₂′ etc. residues ofthe inhibitor. As for the targeting group, the additional targeting,residues are chosen to confer increased specificity on the inhibitor forthe particular cysteine protease to be inhibited.

The choice of the amino acid side chains of the targeting group and ofthe additional targeting residues will be done using the availableinformation about the substrate specificity of the protease, and isroutine to those skilled in the art using commercially availablesubstrates. For example, interleukin-1β converting enzyme displays thegreatest specificity demonstrated for a cysteine protease toward asubstrate, requiring an aspartyl side chain in the P₁ position. Thepapain superfamily of cysteine proteases have an extended specificitysite containing five to seven significant subsites, with the dominantone being S₂, which is a hydrophobic pocket that binds phenylalanyl-likesidechains very well. Cathepsin B, similar to papain, accepts aphenylalanine side chain in S₂, as well as an arginyl sidechain.

For a general review, see “Proteinase Inhibitors”, in ResearchMonographs in Cell and Tissue Physiology (1986), ed. Barrett et al.,Vol. 12, Chapter 4: Inhibitors of Cysteine Proteinases, Daniel Rich,Elsevier, New York, hereby expressly incorporated by reference. Inaddition, the specificity of the interleukin 1β converting enzyme (ICE),was explored in Thornberry et al., supra, also expressly incorporated byreference herein. Table 1 lists some of the favored amino acid sidechains for the “X” (P₂), R₁₁ (P₁), etc. positions for a number ofcysteine proteases.

TABLE 1 enzyme X (P₂) R₁₁ (P₁) papain phe, try, 2-napthyl, leu, arg,lys, lys (ε-Z), nle, ile, ala guanidino-phenylalanine, hph, nlecathepsin B phe, tyr, tyr (I₂), 2- arg, lys, lys (ε-Z), napthyl, arg,guanidino- guanidino-phenylalanaine, phenylalanine, Cit* hph, cit, nlecathepsin L or phe, tyr, 2-napthyl arg, lys, lys (ε-Z), cruzainguanidino-phenylalanaine, hph, cit, nle cathepsin S phe, tyr, 2-napthyl,val, arg, lys, lys (ε-Z), leu, nle, ile, ala guanidino-phenylalanaine,hph, cit, nle DPP-1 gly, ala phe, tyr calpain val, leu, nle, ile, phetyr, phe, met, met (O₂), val ICE ala, val asp *citrulline

The targeting group of the cysteine protease inhibitor may also containadditional functional groups, as shown in Formula 1, above. Thus, theR₁₀ group of Formula 1 may be a hydrogen, a peptide amino end blockinggroup, a peptide residue with or without an amino end blocking group, asingle amino acid residue with or without an amino end blocking group,or a label. In some embodiments, the R₁₀ group may also be a label, suchas a fluorescent label. By the term “peptide amino end blocking group”herein is meant, for example, groups including, but not limited to, analkoxy-ω-oxoalkanoyl of 2 to 10 carbon atoms, alkoxycarbonyl of overall2 to 10 carbon atoms, alkanoyl of overall 2 to 10 carbon atoms,cycloalkylcarbonyl of overall 4 to 8 carbon atoms, carbamoyl,alkylcarbamoyl, or dialkylcarbamoyl, a benzoyl, an alkylsulfonyl ofoverall 1 to 10 carbon atoms, especially alkoxycarbonyl of overall 4 to8 carbon atoms, particularly tert-butoxycarbonyl (BOC) orbenzyloxycarbonyl (CBZ, Z), especially cycloalkylaminocarbonyl oroxacycloalkylaminocarbonyl of overall 4 to 8 atoms in the ring,particularly 4-morpholinecarbonyl (Mu).

The amino acids, or peptide residues, are normally linked via a peptidebond or linkage, i.e. a peptidic carbamoyl group, i.e. —CONH—. Thus, inFormula 1, A=CO and B=NH. However, peptidomimetic bonds are alsoincluded, such as CH₂—NH, CO—CH₂, azapeptide and retro-inverso bonds.

By the term “alkene bond”, or grammatical equivalents, herein is meant acarbon-carbon double bond. It is to be understood that the alkene bondsof the cysteine protease inhibitors confer Michael acceptorfunctionality to the cysteine protease inhibitors. In a preferredembodiment, the carbon to which the R₁₁ amino acid side chain isattached is directly attached to the carbon-carbon double bond.

In a preferred embodiment, the targeting group is linked to one of thecarbons of the carbon-carbon double bond, and the EWG is linked to theother carbon of the carbon-carbon double bond. By the term “linked” orgrammatical equivalents herein is meant a covalent attachment. For thetargeting group linkage to the alkene bond, the linkage may be direct,such that the carbon to which the R₁₁ amino acid side chain is attachedis attached to one of the carbons of the carbon-carbon double bond.Alternatively, there may be other groups between the targeting group andthe alkene bond that do not substantially diminish the ability of thetargeting group to target an enzyme. In the case of the linkage betweenthe alkene bond and the EWG, the linkage is such that the electronwithdrawing properties of the EWG are exerted on the alkene bond, toallow nucleophilic attack by a cysteine protease on the alkene bond.

In a preferred embodiment, the targeting group and the EWG are attachedby the alkene bond in trans configuration. In alternative embodiments,the targeting group and the EWG are attached in cis configuration.(α,β-unsaturated compounds in the cis (Z) configuration, as opposed tothe trans (E) configuration, are made by using phosphonates bearing thehighly electrophilic β-trifluoroethoxy groups at the phsophorus atom(Still et al., Tetrahedron Lett. 24:4405 (1983)) in place ofphosphonates bearing dialkyl groups such as diethyl.

By the term “electron withdrawing group” or “EWG” or grammaticalequivalents herein is meant a functional group that allows nucleophilicattack by the thiol-group of a cysteine protease at the alkene bond ofthe inhibitor as a result of the electron withdrawing properties of theEWG. Thus the EWG is conjugated with the alkene bond, such that theelectron withdrawing properties of the EWG serve to allow nucleophilicattack by a cysteine protease at the alkene bond, i.e. the alkene bondand the EWG are electronically conjugated. Thus, preferably the linkagebetween the alkene bond and the EWG is a direct one, without interveningmoieties that would prevent the electron withdrawing properties of theEWG from being exerted on the alkene bond.

In some embodiments, an EWG comprises an electron withdrawing moiety,EWM, as defined below. In alternative embodiments, an EWG comprises aring (e.g. a five or six membered aromatic ring) that is substitutedwith at least one EWM, a meta directing moiety or group (MDG), or adeactivating group (DG), as defined below. It is to be understood, aswill be described more fully below, that an EWG that contains structuressuch as five or six membered rings between a functional EWM, MDG or DG,and the alkene bond, is permissible as long as the ability of the EWM,MDG or DG to exert an electron withdrawing force on the double bond isnot substantially diminished; i.e. that nucleophilic attack by thecysteine protease at the alkene bond may still occur.

In one embodiment, the EWG comprises a homocyclic six membered aromaticring or a heterocyclic five or six membered aromatic ring, which issubstituted with an EWM, MDG or DG. The ring must be aromatic, that is,it must conform to Huckel's rule for the number of delocalized πelectrons.

If the ring is a six membered homocyclic ring, i.e. containing onlycarbon, it is substituted, with a substitution group, in such a mannerthat the substituted ring, when linked to the alkene bond of a cysteineprotease inhibitor, allows nucleophilic attack by the protease at thealkene bond. Thus the substituted homocyclic ring is an EWG. Thesubstitution group may be an EWM, MDG, or DG.

In one embodiment, a homocyclic ring may have at least one EWM, asdefined below as useful in the Wadsworth-Emmons reaction, substituted atany of the carbons of the ring, to confer electron withdrawingproperties on the EWG. Alternative embodiments have more than one EWMsubstituted on the ring, with up to five substitution groups on a sixmembered aromatic ring.

In alternative embodiments, the substitution group may or may not be anEWM, as defined below, but instead is considered meta directing inrespect to electrophilic aromatic substitution. That is, that theelectron withdrawing properties of the substitution group, when presenton an aromatic ring, direct further addition of groups to the ring to ameta position, relative to the original substitution group. Metadirecting groups or moieties, in respect to electrophilic aromaticsubstitution, are well known in the art. Examples of meta directingmoieties are the quaternary ammonium salts, NR₃+, where R may be forexample an aryl, alkyl or an aralkyl group, among others, as well assuch meta directing groups as NO₂, SO₃H, SO₂R, SOR, SO₂NH₂, SO₂NHR,SO₂NHR₂, SONH₂, SONHR, SONR₂, CN, PO₃H, P(O)(OR)₂, P(O)OR, COOH, COR,and COOR′.

In one embodiment, the substitution group is a deactivating group.“Deactivating group” (DG) or grammatical equivalents, means a group ormoiety which is deactivating in respect to electrophilic aromaticsubstitution, by virtue of their electronegativity, as is well known inthe art. That is, a substitution group that is deactivating renders thesubstituted aromatic ring to be less reactive than the non-substitutedring. In this embodiment, examples of suitable deactivating groups areall halogen atoms, such as F, Cl, Br, I, and At; for example, F₅, CF₃,and (CF₃)_(n).

The substitution may be either in the para, ortho or meta position,relative to the ring carbon that is attached to one of the carbons ofthe carbon-carbon double bond of the cysteine protease inhibitor.

Alternatively, the ring may be heterocyclic aromatic rings, that is,contain more than one kind of atom. Thus five membered rings thatcontain at least one nitrogen, oxygen, phosphorus, arsenic or sulfuratom, and six membered rings that contain at least one nitrogen orphosphorus atom, may be used as outlined below. The rings may containmore than one of the atoms, as well as combinations of atoms. Inaddition, in the preferred embodiment, the heterocyclic five memberedrings may be substituted, as outlined above, with a group that is an EWMas defined below. The six membered rings may be substituted with a groupthat is either an EWM as defined below, a MDG or a DG, as defined above.

Examples of suitable heterocyclic aromatic rings are pyrrole, furan,thiophene, pyridine, thiazole, pyrimidine, phosphole, and arsole.Particularly useful substitution groups for these rings are quaternarypyridinium salts, such as NR₃+, where R is, for example, an aralkyl oralkyl group.

EWGs containing five or six membered aromatic substituted rings usefulin the present invention are determined by their ability to be used inthe Wittig reaction. The Wittig reaction is a well known method ofsynthesizing alkenes from carbonyl compounds, wherein the carbonyloxygen is replaced by a carbon atom, in a reaction with a phosphoniumylide or phosphine oxide. The reaction proceeds if the phosphonium ylideor phosphine oxide contains an electron withdrawing group. Thus anelectron withdrawing group that will function in the Wittig reaction canbe an EWG in the present invention. This reaction is well characterizedand one skilled in the art will be able to choose and assay likely EWGcandidates, using routine techniques.

In a preferred embodiment, the EWG comprises an EWM. In this embodiment,the EWM group is attached to or conjugated directly with one of thecarbons of the carbon-carbon double bond. By the term “electronwithdrawing moiety” or “EWM” or grammatical equivalents herein is meantany functional group that can be used in the Wadsworth-Emmonsmodification of the Wittig reaction in olefin synthesis. Thus, theability of a functional group to form α,β unsaturated hydrocarbons inthe Wadsworth-Emmons reaction will confer the required level of electronwithdrawing to function as an EWG in a cysteine protease inhibitor.

One skilled in the art will be able to routinely test theelectron-withdrawing properties of a functional group using theWadsworth-Emmons modification of the Wittig reaction (see Wadsworth etal., J. Am. Chem. Soc. 83:1733, (1961)). For example, the alkali metalanion of (RO)₂P(O)CH₂—EWG will react with an aldehyde to form the α,βunsaturated alkene.

Preferably, the EWGs of the present invention are vinylogous moieties,that is, they are attached to the carbon-carbon double bond of thecysteine protease inhibitor. As used herein, the term “vinylogous EWG”means an EWG linked to the alkene bond of the inhibitor; that is, thealkene bond is not part of the EWG. Some of the vinylogous compoundsthat are useful in the present invention include, but are not limitedto, vinylogous esters, vinylogous sulfones, vinylogous carboxylates,vinylogous amides, vinylogous phosphonates, vinylogous ketones,vinylogous nitriles, vinylogous sulfoxides, vinylogous sulfonamides,vinylogous sulfinamides, vinylogous nitro compounds, vinylogoussulfonates and vinylogous sulfoximines.

In a preferred embodiment, the cysteine protease inhibitor includes avinylogous ester moiety as the EWG, as shown in Formula 2:

wherein

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid residuewith or without an amino end blocking group or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

R₁ is an ester moiety; and

the A—B linkage is a peptide residue or an isosteric form thereof.

By the term “ester moiety” herein is meant a group including, but notlimited to, an alkyl, a cycloalkyl, a cycloalkylalkyl, an aryl, or anaralkyl. In such an instance, alkyl is preferably of 1 to 5 carbonatoms, especially ethyl. Cycloalkyl preferably is of 3 to 7 carbonatoms, preferably cyclopentyl or cyclohexyl. Cycloalkylalkyl preferablyis of 3 to 7 carbon atoms in the cycloalkyl, particularly 5 or 6 carbonatoms, and of 1 to 5 carbon atoms, particularly 1 carbon atom, in thealkyl moieties thereof. Aryl preferably is phenyl. Aralkyl preferably isphenylalkyl of 7 to 12 carbon atoms, particularly benzyl. The optionalsubstituents of an aryl or aralkyl moiety preferably are one or twogroups alkyl of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms,halogen of atomic number of from 9 to 35, hydroxy and/or amino,preferably one or two groups methyl, methoxy, chlorine, bromine,fluorine, hydroxy or amino, particularly one hydroxy, amino, chlorine,bromine, or fluorine, optionally in protected form where appropriate,nitro, alkyl or arylsulfonyl, or halogen-substituted alkyl of 1 to 5carbon atoms, particularly trifluoromethyl. Particularly preferred R₁groups include C1-C5 alkyl, especially ethyl; C3-C7 cycloalkyl,especially cyclopentyl or cyclohexyl; C3-C7 (cycloalkyl)-C1-C5 alkyl,especially C5-C6(cycloalkyl)-methyl; phenyl; C7-C12 phenylalkyl,especially benzyl; aryl or aralkyl substituted by one or two groups ofC1-C5 alkyl, C1-C5 alkoxy, halogen, hydroxy or amino, with one or twogroups of methyl, methoxy, chlorine, bromine, fluorine, hydroxy or aminobeing preferred and hydroxy, amino, chlorine, bromine or fluorine beingparticularly preferred. Also preferred are aryl or aralkyl substitutedby one or two groups of halogen-substituted C1-C5 alkyl, especiallytrifluoromethyl; nitro; sulfonyl; or arylsulfonyl, in protected formwhere appropriate.

In a specific preferred embodiment, cysteine protease inhibitorscontaining vinylogous esters as EWGs are (1) ethyl(S)-(E)-4-(4-morpholinecarbonylphenylalanyl)-amino-6-phenyl-2-hexenoate,abbreviated herein as Mu-Phe-HphVEOEt, (2) ethyl(S)-(E)-7-guanidino-4-(4-morpholinecarbonylphenylalanyl)amino-2-heptenoatehydrobromide, abbreviated herein as Mu-Phe-ArgVEOEt.HBr; (3)(S)-(E)-Ethyl8-(benzyloxycarbonyl)amino-4-(4-morpholinecarbonylphenylalanyl)amino-2-octenoate,abbreviated herein as Mu-Phe-Lys(z)VEOEt; and (4) (S)-(E)-Ethyl8-amino-4-(4-morpholinecarbonylphenylalanyl)amino-2octenoatehydrobromide, abbreviated herein as Mu-Phe-LysVEOEt-HBr.

In an additional preferred embodiment, the cysteine protease inhibitorincludes a vinylogous sulfone moiety as the EWG, as shown in Formula 3:

wherein

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X an R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

R₂ is a side chain moiety; and

the A—B linkage is a peptide residue or an isosteric form thereof.

The S—R₂ bond is a sulfur-carbon link where R₂ is a side chain moiety.The R₂ side chain moiety may be a group including, but not limited to,an alkyl, a substituted alkyl, a cycloalkyl, a cycloalkylalkyl, acycloalkylalkenyl, an aryl, or an aralkyl. In such an instance, alkyl ispreferably of 1 to 5 carbon atoms, especially methyl. Substituted alkylis preferably of 1 to 5 carbon atoms, bearing substitutions of alkoxy of1 to 5 carbon atoms, halogen of atomic number of from 9 to 35, hydroxyand/or amino, preferably one or two groups methyl, methoxy, chlorine,bromine, fluorine, hydroxy or amino, particularly one hydroxy, amino,chlorine, bromine, or fluorine, optionally in protected form whereappropriate, nitro, alkyl or arylsulfonyl, or halogen-substituted alkylof 1 to 5 carbon atoms, particularly trifluoromethyl. Cycloalkylpreferably is of 3 to 7 carbon atoms, preferably cyclopentyl orcyclohexyl. Cycloalkylalkyl or cycloalkylalkenyl preferably is of 3 to 7carbon atoms in the cycloalkyl, particularly 5 or 6 carbon atoms, and of1 to 5 carbon atoms, particularly I carbon atom, in the alkyl oralkylene moieties thereof. Aryl preferably is phenyl, pentafluorophenylor naphthyl. Aralkyl preferably is phenylalkyl of 7 to 12 carbon atoms,particularly benzyl. The optional substituents of an aryl or aralkylmoiety preferably are one or two groups alkyl of 1 to 5 carbon atoms,alkoxy of 1 to 5 carbon atoms, halogen of atomic number of from 9 to 35,hydroxy and/or amino, preferably one or two groups methyl, methoxy,chlorine, bromine, fluorine, hydroxy or amino, particularly one hydroxy,amino, chlorine, bromine, or fluorine, optionally in protected formwhere appropriate, nitro, alkyl or arylsulfonyl, or halogen-substitutedalkyl of 1 to 5 carbon atoms, particularly trifluoromethyl.

Particularly preferred R₂ groups include (1) C1-C5 alkyl, especiallymethyl; (2) C3-C7 cycloalkyl, preferably cyclopentyl or cycolhexyl; (3)C3-C7(cycloalkyl)-C1-C5 alkyl, preferably C5-C6(cycloalkyl)methyl; (4)C3-C7(cycloalkylalkenyl)-C1-C5 alkyl, preferablyC5-C6(cycloalkylalkenyl)methyl; (5) phenyl, preferably pentafluorophenylor naphtyl; (6) C7-C12 phenylalkyl, preferably benzyl; (7) C1-C5 alkylsubstituted by C1-C5 alkyoxy, halogen, hydroxy or amino, with C1-C5alkyl preferably substituted by one or two groups selected from methyl,methoxy, chlorine, bromine, fluorine, hydroxy or amino, with hydroxy,amino, chlorine, bromine or fluorine being most preferred; (8) C1-C5alkyl substituted with nitro, alkyl or arylsufonyl, optionally protectedwhere appropriate; (9) C1-C5 alkyl substituted with halogen, preferablytrifluoromethyl; (10) aryl or aralkyl substituted by one or two groupsof C1-C5 alkyl, C1-C5 alkoxy, halogen, hydroxy or amino, with one or twogroups of methyl, methoxy, chlorine, bromine, fluorine, hydroxy or aminobeing preferred and hydroxy, amino, chlorine, bromine or fluorine beingparticularly preferred; (11) aryl or aralkyl substituted by one or twogroups of halogen-substituted C1-C5 alkyl, especially trifluoromethyl;nitro; sulfonyl; or arylsulfonyl, in protected form where appropriate.

Preferred cysteine protease inhibitors with vinylogous sulfones as theEWG include: (1)(S)-(E)-5-(4-morpholinecarbonylphenylalanyl)amino-7-phenyl-2-thia-3-heptene2,2-dioxide, abbreviated herein as Mu-Phe-HphVSMe; (2)(S)(E)-3-tert-butoxycarbonylamino-4-methyl-1-phenylsulfonyl-1-pentene,abbreviated herein as Boc-ValVSPh; (3)(S)-(E)-3-(4-morpholinecarbonyl-phenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated herein as Mu-Phe-HphVSPh; (4)(S)-(E)-3-tert-butoxycarbonylamino-4-tert-butoxycarbonyl-1-methylsulfonyl-1-butene,abbreviated herein as (Boc-Asp(Ot-Bu)VSMe; (5)(S)-(E)-3-amino-4-tert-butoxycarbonyl-1-methylsulfonyl-1-butene,abbreviated TsOH.Asp(Ot-Bu)VSMe; (6)(S)-(E)-3-tert-butoxycarbonylamino-4-tert-butoxycarbonyl-1-phenylsulfonyl-1-butene,abbreviated Boc-Asp(Ot-Bu)-VSPh; (7)(S)-(E)-3-amino-4-tert-butoxycarbonyl-1-phenylsulfonyl-1-butene-p-toluenesulfonate,abbreviated TsOH.Asp(Ot-Bu)VSPh; (8)(S)-(E)-3-amino-4-hycroxylcarbonyl-1-phenylsulfonyl-1-butene-p-toluenesulfonate,abbreviated HCl.AspVSPh; (9)(E)-3-acetyltyrosylvalylalanylamino-4-tert-butoxycarbonyl-1-phenylsulfonyl-1-butene,abbreviated Ac-Tyr-Val-Ala-Asp(Ot-Bu)VSPh; (10)(e)-3-acetyltyrosylvalylalanylamino-4-hydroxycarbonyl-1-phenylsulfonyl-1-butene(Ac-Tyr-Val-Ala-AspVSPh; (11)(S)-(E)-3-(4-morpholinecarbonylleucyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Leu-HphVSPh, (12)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-6-guanidino-1-phenylsulfonyl-1-hexenehydrobromide, abbreviated Mu-Phe-ArgVSPh.HBr; (13)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Phe-HphVSPh; (14)(S)-(E)-3-glycylamino-4-phenyl-1-phenylsulfonyl-1-butene hydrochloride,abbreviated Gly-PheVSPh.HCl; (15)(S)-(E)-7-(benzyloxycarbonyl)amino-3-(4-morpholinecarbonylphenylalanyl)-amino-1-phenylsulfonyl-1-heptene,abbreviated Mu-Phe-Lys(Z)VSPh: (16)(G)-(E)-7-amino-3-(4-morpholinecarbonylphenylalanyl)amino-1-phenylsulfonyl-1-heptenehydrobromide, abbreviated Mu-Phe-LysVSPh.HBr; (17)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-4-methyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Phe-ValVSPh; (18)(S)-(E)-3-amino-4-phenyl-1-phenylsulfonyl-1-butene hydrochloride,abbreviated PheVSPh.HCl; (19)(S)-(E)-3-(4-morpholinecarbonylvalyl)amino-4-phenyl-1-phenylsulfonyl-1-butene,abbreviated Mu-Val-PheVSPh; (20)(S)-(E)-3-(4-morpholinecarbonylarginyl)amino-6-guanidino-1-phenylsulfonyl-1-hexenedihydrobromide, abbreviated Mu-ARg-Arg-VSPh.2HBr; (21)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-4-benzyloxy-1-phenylsulfonyl-1-butene,abbreviated Mu-Phe-SEr(OBzl)VSPh; (22)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-7-benzoylamino-1-phenylsulfonyl-1-heptene,abbreviated Mu-Phe-Lys(Bz)VSPh; (23)(R)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Phe-D-HphVSPh; (24)(S)-(E)-3-[4-morpholinecarbonyl-(3,5-diiodotyrosyl)]-amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Tyr(I₂)-HphVSPh; (25)(S)-(E)-3-[4-tert-butoxycarbonyl-(3,5-diiodotyrosyl)]-amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Boc-Tyr(I₂)-HphVSPh; (26)(S,S)-(E)-3-[4-morpholinecarbonyl-(1,2,3,4-tetrahydro-3-isoquinoline-carbonyl)]amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Tic-HphVSPh; (27)(S,S)-(E)-3-[tert-butoxycarbonyl-(1,2,3,4-tetrahydro-3-isoquinolinecarbonyl)]-amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Boc-Tic-HphVSPh; (28)(S)-(E)-3-(4-morpholinecarbonylleucylleucyl)amino-4-(4-hydroxyphenyl)-1-phenylsulfonyl-1-butene,abbreviated Mu-Leu-HphVSPh; (29)(S)-(E)-3-amino-5-phenyl-1-phenylsulfonyl-1-pentene hydrochloride,abbreviated HphVSPh-HCl; (30)(S)-(E)-3-[(4-morpholinecarbonyl-(R,S)-α-methylphenylalanyl]amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-DL-Fam-HphVSPh; (31)(S)-(E)-3-(benzyloxycarbonylleucylleucyl)amino-4-(4-hydroxyphenyl)-1-phenylsulfonyl-1-butene,abbreviated Z-Leu-Leu-TyrVSPh; (32)(S)-(E)-3-(4-morpholinecarbonyltyrosyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Tyr-HphVSPh; (33)(S)-(E)-3-(tert-butoxycarbonyl-2-naphthylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Boc-Np-HphVSPh; (34)(S)-(E)-3-(4-morpholinecarbonyl-2-naphthylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Np-HphVSPh; (35)(S)-(E)-3-(4-morpholinecarbonyl-4-biphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Bip-HphVSPh; (36)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-phenylsulfonyl-1-heptene,abbreviated Mu-Phe-NleVSPh; (37)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-phenylsulfonyl-6-thia-1-heptene,abbreviated Mu-Phe-MetVSPh; (38)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-5-methylsulfonyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Phe-Met(O₂)VSPh; (39)(S)-(E)-3-(acetylleucylleucyl)amino-1-phenylsulfonyl-1-heptene,abbreviated Ac-Leu-Leu-NleVSPh; (40)(S)-(E)-3-(acetylleucylleucyl)amino-1-phenylsulfonyl-6-thia-1-heptene,abbreviated Ac-Leu-Leu-MetVSPh; (41)(S)-(E)-3-(acetylleucylleucyl)amino-5-methylsulfonyl-1-phenylsulfonyl-1-pentene,abbreviated Ac-Leu-Leu-Met(O₂)VSPh; (42)(S)-(E)-3-(carbomethoxypropionylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated MeOSuc-Phe-HphVSPh; (43)(S)-7-(benzyloxycarbonyl)amino-3-(4-morpholinecarbonylphenylalanyl)amino-1-fluoro-1-phenylsulfonyl-1-heptene,abbreviated Mu-Phe-Lys(Z)fVSPh; (44)(S)-(E)-3-(acetylleucylleucyl)amino-4-(4-hydroxyphenyl)-1-phenylsulfonyl-1-butene,abbreviated Ac-Leu-Leu-TyrVSPh; (45)(S)-(E)-3-(dimethylsulfamoylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated DimSam-Phe-HphVSPh; (46)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-(4-bromophenylsulfonyl)-5-phenyl-1-pentene,abbreviated Mu-Phe-HphVSPhBr; (47)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-(2-napthylsulfonyl)-5-phenyl-1-pentene,abbreviated Mu-Phe-HphVSNp; (48)(S)-(E)-3-(4-morpholinecarbonyl-2-naphthylalanyl)amino-1-(2-napthylsulfonyl)-5-phenyl-1-pentene,abbreviated Mu-Np-HphVSNp; (49)(S)-(E)-3-(4-morpholinecarbonyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-HphVSPh; (50)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-methylsulfonyl-1-butene,abbreviated Mu-Phe-AlaVSMe; (51)(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-methylsulfonyl-4-phenyl-1-butene,abbreviated Mu-Phe-PheVSMe; and (52)(S)-(E)-3-(tert-2butoxycarbonylalanyl)amino-1-methylsulfonyl-4-phenyl-1-butene,abbreviated Boc-Ala-PheVSMe.

In one embodiment, the cysteine protease inhibitors of the presentinvention include a vinylogous carboxylate as the EWG, as shown inFormula 4:

wherein

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration; and

the A—B linkage is a peptide residue or an isosteric form thereof.

In the preferred embodiment, a cysteine protease inhibitor with avinylogous carboxylate as the EWG is(S)-(E)-4-(4-morpholinecarbonylphenylalanyl)amino-6- phenyl-2-hexenoicacid, abbreviated herein as Mu-Phe-HphVA, and (S)-(E)-Benzyl4-(4-morpholinecarbonylphenylalanyl)amino-6-phenyl-2-hexenamide,abbreviated herein as Mu-Phe-HphVAMbzl.

In a preferred embodiment, the cysteine protease inhibitor includes avinylogous phosphonate as the EWG, as shown in Formula 5:

wherein

R₁₀ hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

R₃ is an ester moiety; and

the A—B linkage is a peptide residue or an isosteric form thereof.

The R₃ groups are ester moieties, and may be groups including, but notlimited to, an alkyl, a cycloalkyl, a cycloalkylalkyl, acycloalkylalkenyl, an aryl, or an aralkyl. In such an instance, alkyl ispreferably of 1 to 5 carbon atoms, especially ethyl. Cycloalkylpreferably is of 3 to 7 carbon atoms, preferably cyclopentyl orcyclohexyl. Cycloalkylalkyl or cycloalkylalkenyl preferably is of 3 to 7carbon atoms in the cycloalkyl, particularly 5 or 6 carbon atoms, and of1 to 5 carbon atoms, particularly 1 carbon atom, in the alkyl oralkylene moieties thereof. Aryl preferably is phenyl. Aralkyl preferablyis phenylalkyl of 7 to 12 carbon atoms, particularly benzyl. Theoptional substituents of an aryl or aralkyl moiety preferably are one ortwo groups alkyl of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms,halogen of atomic number of from 9 to 35, hydroxy and/or amino,preferably one or two groups methyl, methoxy, chlorine, bromine,fluorine, hydroxy or amino, particularly one hydroxy, amino, chlorine,bromine, or fluorine, optionally in protected form where appropriate,nitro, alkyl or arylsulfonyl, or halogen-substituted alkyl of 1 to 5carbon atoms, particularly trifluoromethyl. Also included are perfluorogroups, such as perfluoro alkyl, aryl and aralkyl groups.

Particularly preferred are: (1) C1-C5 alkyl, especially ethyl; (2) C3-C7cycloalkyl, preferably cyclopentyl or cycolhexyl; (3)C3-C7(cycloalkyl)-C1-C5 alkyl, preferably C5-C6(cycloalkyl)methyl; (4)C3-C7(cycloalkylalkenyl)-C1-C5 alkyl, preferablyC5-C6(cycloalkylalkenyl)methyl; (5) phenyl; (6) C7-C12 phenylalkyl,preferably benzyl; (7) C1-C5 alkyl substituted by C1-C5 alkyoxy,halogen, hydroxy or amino, with C1-C5 alkyl preferably substituted byone or two groups selected from methyl, methoxy, chlorine, bromine,fluorine, hydroxy or amino, with hydroxy, amino, chlorine, bromine orfluorine being most preferred; (8) C1-C5 alkyl substituted with nitro,alkyl or arylsufonyl, optionally protected where appropriate; (9) C1-C5alkyl substituted with halogen, preferably trifluoromethyl; (10) aryl oraralkyl substituted by one or two groups of C1-C5 alkyl, C1-C5 alkoxy,halogen, hydroxy or amino, with one or two groups of methyl, methoxy,chlorine, bromine, fluorine, hydroxy or amino being preferred andhydroxy, amino, chlorine, bromine or fluorine being particularlypreferred; (11) aryl or aralkyl substituted by one or two groups ofhalogen-substituted C1-C5 alkyl, especially trifluoromethyl; nitro;sulfonyl; or arylsulfonyl, in protected form where appropriate.

In a preferred embodiment, a cysteine protease inhibitor with avinylogous phosphonate as the EWG is diethyl(S)-(E)-4-(4-morpholinecarbonylphenylalanyl)amino-6-phenyl-2-hexenephosphonate,abbreviated herein as Mu-Phe-HphVPEt.

In another preferred embodiment, the cysteine protease inhibitors of thepresent invention include vinylogous amides as the EWG, as shown inFormula 6:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X, R₁₁ and Z are amino acid side chains, with either (R) or (S)stereochemical configuration,

Q=hydrogen, an ester, a peptide residue, or an amide moiety; and

The A—B linkage is a peptide residue or an isosteric form thereof.

By “amide moiety” as used with regard to Q is meant a group including,but not limited to, an NH2, or an NH-alkyl, an NH-cycloalkyl, anNH-cycloalkylalkyl, an NH-aryl, or an NH-aralkyl, or an N-dialkyl,N-dicycloalkyl, an N-dicycloalkylalkyl, an N-diaryl, or an N-diaralkyl.In such an instance, alkyl is preferably of 1 to 5 carbon atoms,especially ethyl. Cycloalkyl preferably is of 3 to 7 carbon atoms,preferably cyclopentyl or cyclohexyl. Cycloalkylalkyl preferably is of 3to 7 carbon atoms in the cycloalkyl, particularly 5 or 6 carbon atoms,and of 1 to 5 carbon atoms, particularly 1 carbon atom, in the alkylmoieties thereof. Aryl preferably is phenyl. Aralkyl preferably isphenylalkyl of 7 to 12 carbon atoms, particularly benzyl. The optionalsubstituents of an aryl or aralkyl moiety preferably are one or twogroups alkyl of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms,halogen of atomic number of from 9 to 35, hydroxy and/or amino,preferably one or two groups methyl, methoxy, chlorine, bromine,fluorine, hydroxy or amino, particularly one hydroxy, amino, chlorine,bromine, or fluorine, optionally in protected form where appropriate,nitro, alkyl or arylsulfonyl, or halogen-substituted alkyl of 1 to 5carbon atoms, particularly trifluoromethyl.

Particularly preferred for Q are: (1) NH—(C1-C5) alkyl, especiallyNH-ethyl; (2) NH—(C3-C7)cycloalkyl, preferably cyclopentyl orcyclohexyl; (3) HN—(C3-C7)cycloalkyl-(C1-C5) alkyl, preferablyNH—(C5-C6)cycloalkyl-methyl; (4) NH-aryl, preferably NH-phenyl; (5)NH—(C—C12)phenylalkyl, preferably benzyl; (6) aryl or aralkylsubstituted by one or two groups of (C1-C5) alkyl, (preferably methyl),(C1-C5)alkyoxy, (preferably methoxy), or halogen, (preferablychlorine,bromine, fluorine, hydroxy, or amino).

Especially preferred is to substitue the aryl or aralkyl with one groupselected from hydroxy, amino, chlorine, bromine, or fluorine, optionallyin protected form where appropriate, nitro, alkyl or arylsufonyl, orhalogen-substituted (C1-C5)alkyl, especially trifluoromethyl.

The ester as used with regard to Q may be an oxygen linked with theester moieties defined for vinylogous esters and phosphonates to formesters. Specific preferred embodiments, include the following cysteineprotease inhibitors that contain vinylogous amides as the EWG:(S)-(E)-(N-leucylprolinemethylester)-4-(4-morpholinecarbonylphenyl-alanyl)amino-6-phenyl-2-hexenamide,abbreviated herein as Mu-Phe-HphVAM-Leu-ProOMe;(S)-(E)-(N-phenylalanine)-4-(4-morpholinecarbonylphenylalanyl)amino-6-phenyl-2-hexenamide,abbreviated herein as Mu-Phe-HphVAM-PheOH;(S)-(E)-3-(4-morpholinecarbonyl-phenylalanylamino)-6-phenyl-2-hexenamide,abbreviated Mu-Phe-HphVAM; and (S)-(E)-Benzyl4-(4-morpholine-carbonylphenylalanyl)amino-6-phenyl-2-hexenamide,abbreviated Mu-Phe-HphVAMBzl.

Although not preferred, in one embodiment, the cysteine proteaseinhibitor includes a vinylogous ketone as the EWG, as shown in Formula7:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

R₄=ketone moiety; and

the A—B linkage is a peptide residue or an isosteric form thereof.

By “ketone moiety” as used with regard to R₄ herein is meant groupsincluding, but not limited to, an alkyl, a cycloalkyl, acycloalkylalkyl, a cycloalkylalkenyl, an aryl, or an aralkyl. In such aninstance, alkyl is preferably of 1 to 5 carbon atoms, especially ethyl.Cycloalkyl preferably is of 3 to 7 carbon atoms, preferably cyclopentylor cyclohexyl.

Cycloalkylalkyl or cycloalkylalkenyl preferably is of 3 to 7 carbonatoms in the cycloalkyl, particularly 5 or 6 carbon atoms, and of 1 to 5carbon atoms, particularly 1 carbon atom, in the alkyl or alkylenemoieties thereof. Aryl preferably is phenyl. Aralkyl preferably isphenylalkyl of 7 to 12 carbon atoms, particularly benzyl. The optionalsubstituents of an aryl or aralkyl moiety preferably are one or twogroups alkyl of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms,halogen of atomic number of from 9 to 35, hydroxy and/or amino,preferably one or two groups methyl, methoxy, chlorine, bromine,fluorine, hydroxy or amino, particularly one hydroxy, amino, chlorine,bromine, or fluorine, optionally in protected form where appropriate,nitro, alkyl or arylsulfonyl, or halogen-substituted alkyl of 1 to 5carbon atoms, particularly trifluoromethyl. Also included are perfluorogroups, such as perfluoro alkyl, aryl, and aralkyl.

Particularly preferred are: (1) (C1-C5)alkyl, preferably ethyl; (2)(C3-C7)cycloalkyl, preferably cyclopentyl or cyclohexyl; (3)(C3-C7)cycloalkyl-(C1-C5)alkyl, especially (C5-C6)cycloalkyl-methyl; (4)(C3-C7)cycloalkyl-(C1-C5)alkenyl, especially(C5-C6)cycloalkyl-methylene; (5) phenyl; (6) (C7-C12)penylalkyl,especially benzyl; (7) aryl or aralkyl substituted by one or two groupsof C1-C5 alkyl, C1-C5 alkoxy, halogen, hydroxy or amino, with one or twogroups of methyl, methoxy, chlorine, bromine, fluorine, hydroxy or aminobeing preferred and hydroxy, amino, chlorine, bromine or fluorine beingparticularly preferred; (11) aryl or aralkyl substituted by one or twogroups of halogen-substituted C1-C5 alkyl, especially trifluoromethyl;nitro; sulfonyl; or arylsulfonyl, in protected form where appropriate;(12) perfluoro groups, such as perfluoro alkyl, aryl, and aralkyl.

In another preferred embodiment, the cysteine protease inhibitors of thepresent invention include a nitrile group as the EWG, as shown inFormula 8:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ amino acid side chains, with either (R) or (S) stereochemicalconfiguration;

the C≡N group is a nitrile; and

the A—B linkage is a peptide residue or an isosteric form thereof.

In another preferred embodiment, the cysteine protease inhibitors of thepresent invention contain a vinylogous sulfoxide as the EWG, as shown inFormula 9:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

R₅=sulfoxide moiety; and

the A—B linkage is a peptide residue or an isosteric form thereof.

By “sulfoxide moiety” as used with regard to R₅ herein is meant groupsincluding, but are not limited to, an alkyl, a cycloalkyl; acycloalkylalkyl, a cycloalkylalkenyl, an aryl, or an aralkyl. In such aninstance, alkyl is preferably of 1 to 5 carbon atoms, especially ethyl.Cycloalkyl preferably is of 3 to 7 carbon atoms, preferably cyclopentylor cyclohexyl. Cycloalkylalkyl or cycloalkylalkenyl preferably is of 3to 7 carbon atoms in the cycloalkyl, particularly 5 or 6 carbon atoms,and of 1 to 5 carbon atoms, particularly 1 carbon atom, in the alkyl oralkylene moieties thereof. Aryl preferably is phenyl. Aralkyl preferablyis phenylalkyl of 7 to 12 carbon atoms, particularly benzyl. Theoptional substituents of an aryl or aralkyl moiety preferably are one ortwo groups alkyl of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms,halogen of atomic number of from 9 to 35, hydroxy and/or amino,preferably one or two groups methyl, methoxy, chlorine, bromine,fluorine, hydroxy or amino, particularly one hydroxy, amino, chlorine,bromine, or fluorine, optionally in protected form where appropriate,nitro, alkyl or arylsulfonyl, or halogen-substituted alkyl of 1 to 5carbon atoms, particularly trifluoromethyl. Also included are perfluorogroups such as perfluoro alkyl, aryl, and aralkyl.

Particularly preferred for R₅ are:(1) (C1-C5)alkyl, preferably ethyl;(2) (C3-C7)cycloalkyl, preferably cyclohexyl or cyclopentyl; (3)(C3-C7)cycloalkyl-(C1-C5)alkyl, preferably(C5-C6)cycloalklyl-C1alkyl-C1alkyl; (4) (C3-C7)cycloalkyl-(C1-C5)alkenyl, preferably (C5-C6)cycloalkyl-C1alkenyl; (5)phenyl; (6) (C7-C12)phenylalkyl, preferably benzyl; (7) aryl or aralkylsubstituted by one or two groups of C1-C5 alkyl, C1-C5 alkoxy, halogen,hydroxy or amino, with one or two groups of methyl, methoxy, chlorine,bromine, fluorine, hydroxy or amino being preferred and hydroxy, amino,chlorine, bromine or fluorine being particularly preferred; or (8) arylor aralkyl substituted by one or two groups of halogen-substituted C1-C5alkyl, especially trifluoromethyl.

In a preferred embodiment, the EWG of the cysteine protease inhibitorincludes a vinylogous sulfonamide as the EWG, as shown in Formula 10:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X, R₁₁ and Z are amino acid side chains, with either (R) or (S)stereochemical configuration;

Q=hydrogen, an ester, a peptide residue or an amide moiety; and

the A—B linkage is a peptide residue or an isosteric form thereof.

By “amide moiety” as used with regard to Q is meant a group including,but not limited to, an NH2, or an NH-alkyl, an NH-cycloalkyl, anNH-cycloalkylalkyl, an NH-aryl, or an NH-aralkyl, or an N-dialkyl,N-dicycloalkyl, an N-dicycloalkylalkyl, an N-diaryl, or an N-diaralkyl.In such an instance, alkyl is preferably of 1 to 5 carbon atoms,especially ethyl. Cycloalkyl preferably is of 3 to 7 carbon atoms,preferably cyclopentyl or cyclohexyl. Cycloalkylalkyl preferably is of 3to 7 carbon atoms in the cycloalkyl, particularly 5 or 6 carbon atoms,and of 1 to 5 carbon atoms, particularly 1 carbon atom, in the alkylmoieties thereof. Aryl preferably is phenyl. Aralkyl preferably isphenylalkyl of 7 to 12 carbon atoms, particularly benzyl. The optionalsubstituents of an aryl or aralkyl moiety preferably are one or twogroups alkyl of 1 to 5 carbon atoms, alkoxy of 1 to 5 carbon atoms,halogen of atomic number of from 9 to 35, hydroxy and/or amino,preferably one or two groups methyl, methoxy, chlorine, bromine,fluorine, hydroxy or amino, particularly one hydroxy, amino, chlorine,bromine, or fluorine, optionally in protected form where appropriate,nitro, alkyl or arylsulfonyl, or halogen-substituted alkyl of 1 to 5carbon atoms, particularly trifluoromethyl.

By “ester” as regards to Q means an oxygen attached to the ester moitiespreviously defined, to form an ester.

In a preferred embodiment, the cysteine protease inhibitors of thepresent invention include a vinylogous sulfoximine as the EWG, as shownin Formula 11:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

R₆ and R₇ a re sulfoximine moieties; and

the A—B linkage is a peptide residue or an isosteric form thereof.

By “sulfoximine moieties” as used with regard to R₆ and R₇ is meantidentical or different groups including, but not limited to, an alkyl, acycloalkyl, a cycloalkylalkyl, a cycloalkylalkenyl, an aryl, or anaralkyl. In such an instance, alkyl is preferably of 1 to 5 carbonatoms, especially ethyl. Cycloalkyl preferably is of 3 to 7 carbonatoms, preferably cyclopentyl or cyclohexyl. Cycloalkylalkyl orcycloalkylalkenyl preferably is of 3 to 7 carbon atoms in thecycloalkyl, particularly 5 or 6 carbon atoms, and of 1 to 5 carbonatoms, particularly 1 carbon atom, in the alkyl or alkylene moietiesthereof. Aryl preferably is phenyl. Aralkyl preferably is phenylalkyl of7 to 12 carbon atoms, particularly benzyl. The optional substituents ofan aryl or aralkyl moiety preferably are one or two groups alkyl of 1 to5 carbon atoms, alkoxy of 1 to 5 carbon atoms, halogen of atomic numberof from 9 to 35, hydroxy and/or amino, preferably one or two groupsmethyl, methoxy, chlorine, bromine, fluorine, hydroxy or amino,particularly one hydroxy, amino, chlorine, bromine, or fluorine,optionally in protected form where appropriate, nitro, alkyl orarylsulfonyl, or halogen-substituted alkyl of 1 to 5 carbon atoms,particularly trifluoromethyl. Also included are perfluoro groups, suchas perfluoro aryl, alkyl, and aralkyl groups.

Particularly preferred for R₆ and R₇ are: (1) (C1-C5)alkyl, preferablyethyl; (2) (C3-C7)cycloalkyl, preferably cyclohexyl or cyclopentyl; (3)(C3-C7)cycloalkyl-(C1-C5)alkyl, preferably (C5-C6)cycloalkyl-C1alkyl;(4) (C3-C7)cycloalkyl-(C1-C5)alkenyl, preferably(C5-C6)cycloalkyl-C1alkenyl; (5) phenyl; (6) (C7-C12)phenylalkyl,preferably benzyl; (7) aryl or aralkyl substituted by one or two groupsof C1-C5 alkyl, C1-C5 alkoxy, halogen, hydroxy or amino, with one or twogroups of methyl, methoxy, chlorine, bromine, fluorine, hydroxy or aminobeing preferred and hydroxy, amino, chlorine, bromine or fluorine beingparticularly preferred; or (8) aryl or aralkyl substituted by one or twogroups of halogen-substituted C1-C5 alkyl, especially trifluoromethyl.

In one embodiment, the cysteine protease inhibitors of the presentinvention have the structure shown in Formula 12:

wherein

R₈=the five or six membered homo- or heterocyclic aromatic rings with atleast one substituted EWM, MDG, or DG; and

R₉=a suitable group as defined below.

The R₈ group is an EWG group. It may be a five or six memberedhomocyclic aromatic ring with at least one substitution group. Thesubstituted group may be an EWM, as defined above, in the case of fivemembered rings, or an EWM, MDG, or DG as defined above. EWMs that may besubstituted onto a five or six membered ring that are preferred areesters, sulfones, carboxylates, amides, phosphonates, ketones, nitrites,nitro compounds, sulfonates, sulfoxides, sulfonamides, sulfinamides, andsulfoximines, as are defined above. MDGs that may be used are thequaternary ammonium salts, NR₃+, where R may be for example an aryl,alkyl or aralkyl, as well as such meta directing groups as NO₂, SO₃H,SO₂R, SOR, SO₂NH₂, SO₂NHR, SO₂NHR₂, SONH₂, SONHR, SONR₂, CN, PO₃H,P(O)(OR)₂, P(O)OR, OH, COOH, COR, and COOR′. DGs that may be substitutedonto a six membered ring are all the halogen atoms, such as F, Cl, Br,I, and At; for example, F₅, CF₃, and (CF₃)_(n).

The R₉ group may be a wide variety of groups. In the preferredembodiment, the R₉ group is a hydrogen atom, or other functionallyneutral groups such as methyl groups. In alternative embodiments, the R₉group is a peptide, such that additional targeting orspecificity-enhancing residues are added to the cysteine proteaseinhibitor.

In a preferred embodiment, the cysteine protease inhibitors of thepresent invention have a vinylogous five membered heterocyclic aromaticring with a substituted EWM, as the EWG, as shown in Formula 13:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

the A—B linkage is a peptide residue or an isosteric form thereof;

D=an oxygen, sulfur, nitrogen, phosphorus or arsenic atom; and

EWM=an electron withdrawing moiety.

The EWM may be any of the electron withdrawing groups as previouslydefined.

In a preferred embodiment, the cysteine protease inhibitors of thepresent invention have a vinylogous six membered homocyclic aromaticring with a substituted EWM, MDG or DG as the EWG, as shown in Formula14:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

the A—B linkage is a peptide residue or an isosteric form thereof;

EWM=an electron withdrawing moiety;

MDG=a meta directing group; and

DG=a deactivating group.

In a preferred embodiment, the cysteine protease inhibitors of thepresent invention have a vinylogous six membered heterocyclic aromaticring with a substituted EWM as the EWG, as shown in Formula 15:

wherein

R₁₀=hydrogen, a peptide amino end blocking group, a peptide residue withor without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

the A—B linkage is a peptide residue or an isosteric form thereof;

T=a nitrogen or phosphorus atom; and

EWM=an electron withdrawing moiety.

The EWM may also be attached to the “T” heteroatom.

In a further embodiment, the targeting group and the EWG are linked by adiene bond. By “diene bond” herein is meant a chain of four carbon atoms(C1, C2, C3, and C4) in which C1 and C2 are connected by a double bond,C3 and C4 are connected by a double bond, and C2 and C3 are connected bya single bond. In this embodiment, the targeting group is linked to thefirst carbon of the first carbon-carbon double bond (C1), and the EWG islinked to the last carbon of the second carbon-carbon double bond (C4).In a preferred embodiment, the two double bonds of the diene are in(E)-(E) configuration, although alternative embodiments utilize (E)-(Z),(Z)-(E), or (E)-(E) configurations.

In a preferred embodiment, the second order rate constant for inhibitionof a cysteine protease with the diene bond cysteine protease inhibitor,expressed as k_(irr)K_(I), is at least about 1000 M⁻¹sec⁻¹, withalterative embodiments having second order rate constants at least about10,000 M⁻¹sec⁻¹, with the most preferred rate constant being at leastabout 100,000 M⁻¹sec⁻¹.

In this embodiment, the targeting group and EWG are defined as above.

In a preferred embodiment, the cysteine protease inhibitor comprises acompound with the formula:

wherein

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

A—B is a peptide linkage; and

EWG is an electron withdrawing group.

In an additional embodiment, the cysteine protease inhibitors of thepresent invention comprise a targeting group linked via an alkene bondto two EWGs. In this embodiment, the targeting group is linked to one ofthe carbons of the carbon-carbon double bond, and the two EWGs arelinked to the other carbon of the carbon-carbon double bond. The linkageof the targeting group to the alkene bond is as described above for thesingle EWG embodiment The linkage of the two EWGs to the alkene bond issimilar as for one EWG; that is, the linkage is such that the electronwithdrawing properties of the EWGs are exerted on the alkene bond, toallow nucleophilic attack by a cysteine protease on the alkene bond.

In this embodiment, preferably the two EWGs are different. In analternative embodiment, the two EWGs are the same.

In a preferred embodiment, the cysteine protease inhibitor comprises acompound with the formula:

wherein

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label;

X and R₁₁ are amino acid side chains, with either (R) or (S)stereochemical configuration;

A—B is a peptide linkage; and

EWG is an electron withdrawing group.

In one embodiment, the cysteine protease inhibitors are comprised of atargeting group linked to an alkene bond conjugated with an electronwithdrawing group (EWG).

In this embodiment, the second order rate constant for inhibition of acysteine protease with the inhibitor, expressed as k_(irr)/K_(I), is atleast about 1000 M⁻¹sec⁻¹.

By the term “second order rate constant” or grammatical equivalentsherein is meant the kinetic constant associated with a bimolecularreaction that is dependent on the concentration of two species. Thesecond order rate constants are defined and determined as below.

The determination of second order rate constants is known in the art.For example, for irreversible inhibition reactions such as those of thepresent invention, the reaction scheme is as follows:

The reaction is divided into two processes. The enzyme and the inhibitorfirst combine to give an enzyme-inhibitor complex, E.I. This step isassumed to be rapid and reversible, with no chemical changes takingplace; the enzyme and the inhibitor are held together by non-covalentforces. In this reaction, k₁ is the second order rate constant for theformation of the E.I reversible complex. k₂ is the first order rateconstant for the disassociation of the reversible EI complex. Thechemical processes then occur in a second step with a first order rateconstant k_(irr), which is the constant for the inactivation of the E.Icomplex, which is similar to the k_(cat) constant or turnover number. Inthis reaction, K_(I)=k₂ ₁k₁, and the second order rate constant ofinactivation is k_(irr)/K_(I).

The measurement of the first order rate constant k_(irr) and theequilibrium constant K_(I) proceeds according to techniques well knownin the art, as described in the examples. For example, assays are run inthe presence of inhibitor and substrate, generally using syntheticchromogenic substrates. Inhibition progress curve analysis usingnon-linear curve fitting software widely available enables thecalculation of the first order rate constant k_(irr) and the equilibriumconstant K_(I). The second order rate constant, k_(irr)/K_(I), is thencalculated as follows:

At equilibrium, k₁[E][I]=k₂[E.I]

or$\frac{\lbrack E\rbrack \lbrack I\rbrack}{\left\lbrack {E \cdot I} \right\rbrack} = {\frac{k_{2}}{k_{1}} = K_{I}}$

where K_(I) is a dissociation constant with units of M.

For the inactivation of E,${{\frac{}{t}E} - I} = {v_{E - I} = {k_{irr}\left\lbrack {E \cdot I} \right\rbrack}}$E_(total) = E + E ⋅ I$K_{I} = \frac{\lbrack E\rbrack \lbrack I\rbrack}{\left\lbrack {E \cdot I} \right\rbrack}$$\frac{v_{E - I}}{\left\lbrack E_{T} \right\rbrack} = \frac{k_{irr}\left\lbrack {E \cdot I} \right\rbrack}{\lbrack E\rbrack + \left\lbrack {E \cdot I} \right\rbrack}$$\frac{v_{E - I}}{\left\lbrack E_{T} \right\rbrack} = \frac{k_{irr}\quad \frac{\lbrack E\rbrack \lbrack I\rbrack}{K_{I}}}{\lbrack E\rbrack + \frac{\lbrack E\rbrack \lbrack I\rbrack}{K_{I}}}$

Eliminating [E]:$\frac{v_{E - I}}{\left\lbrack E_{T} \right\rbrack} = \frac{k_{irr}\quad \frac{\lbrack I\rbrack}{K_{I}}}{1 + \frac{I}{K_{I}}}$

Using K_(I) as a common denominator gives:$\frac{v_{E - I}}{\left\lbrack E_{T} \right\rbrack} = \frac{k_{irr}\quad\lbrack I\rbrack}{K_{I} + \lbrack I\rbrack}$

and:$v_{E - I} = \frac{{k_{irr}\quad\lbrack I\rbrack}\left\lbrack E_{T} \right\rbrack}{\lbrack I\rbrack + K_{I}}$

k_(irr) is the first order rate constant of inactivation of E. When[I]<<K_(I), the rate equation reduces to$v_{E - I} = {{\frac{k_{irr}\quad}{K_{I}}\quad\lbrack I\rbrack}\left\lbrack E_{T} \right\rbrack}$

that is the pseudo-second order rate expression where k_(irr)/K_(I)represents the pseudo-second order rate constant with units of M⁻¹sec⁻¹.

It is to be understood that second order rate constants are aparticularly useful way of quantifying the efficiency of an enzyme witha particular substrate or inhibitor, and are frequently used in the artas such. The efficiency of an inhibitor depends on the second order rateconstant and not on either the K_(I) value alone or the k_(irr) valuealone. Thus even if an inhibitor exhibits a very low K_(I), oralternatively a very high k_(irr), it may not be a efficient inhibitorif the second order rate constant is low. Accordingly, the cysteineprotease inhibitors of the present invention have second order rateconstants, expressed as k_(irr)/K_(I), of at least about 1000 M⁻¹sec⁻¹.Preferred embodiments have inhibitors that exhibit second order rateconstants of at least about 10,000 M⁻¹sec⁻¹, with the most preferredembodiments having second order rate constants of at least about 100,000M⁻¹sec⁻¹. In addition, the second order rate constants of the preferredembodiment do not exceed the diffusion limit of about 1×10⁸ M⁻¹sec⁻¹.

In the preferred embodiment, the cysteine protease inhibitors arechiral. In this embodiment, the chiral cysteine protease inhibitor iscomprised of a targeting group linked to an alkene bond conjugated withan EWG. By the term “chiral” or grammatical equivalents herein is meanta compound that exhibits dissymetry. That is, the chiral compound is notidentical with its mirror image. Thus in the preferred embodiment, thecompounds of the present invention are pure epimers. Chiral compounds,and particularly chiral cysteine protease inhibitors, are useful in thepresent invention because biological systems, and enzymes in particular,are stereospecific, preferring the (S) or L-form of amino acids. Thus inthe preferred embodiment, the chiral cysteine protease inhibitors of thepresent invention will have amino acid side chains in the (S) orL-configuration, although some inhibitors may utilize amino acid sidechains in the (R) or D-configuration.

In alternative embodiments, the compositions of the present inventionare not pure epimers, but are mixtures that contain more than 50% of oneepimer. Preferred embodiments have greater than about 70% of one epimer,and the most preferred embodiment has at least about 90% of one epimer.

The synthesis of the cysteine protease inhibitors of the presentinvention proceeds as follows. In general, preparation of the inhibitorsdescribed herein requires first that the amino acids from which theinhibitory functionalities are derived, i.e. the targeting amino acids,be converted into t-butoxycarbonyl-protected a-aminoaldehydes, alsocalled N-protected α-amino aldehydes, as shown in Equation 5 (forexample by the method of Fehrentz, J-A. and Castro, B. (Synthesis,(1983), 676 (Equation 5)).

wherein

R=an amino acid side chain: alkyl, aryl, aminoalkyl, etc.;

a) Cl—H₂N+(Me)OMe, dicyclohexylcarbodiimide, triethylamine; and

b) lithium aluminum hydride.

The resulting aldehydes are transformed into vinylogous compounds usingthe Wadsworth-Emmons-Horner modification of the Wittig reaction to makecysteine protease inhibitor intermediates (Wadsworth et al., J. Amer.Chem. Soc. 83:1733 (1961); Equation 1).

In one embodiment, the vinylogous compounds, as cysteine proteaseinhibitor intermediates, may be used as cysteine protease inhibitorswithout further chemical addition, i.e. the inhibitors have only asingle amino acid side chain, as depicted in Formula 16:

wherein

R₁₂ is hydrogen or a peptide amino end blocking group;

R₁₁ any amino acid side chain except glycine (hydrogen), or, when theEWG is a vinylogous ester, phenylalanine (benzyl); and

EWG is an electron withdrawing group.

For example, Boc-AspVSPh may be used as an inhibitor of ICE withoutfurther addition of amino acid side chain targeting groups.

In a preferred embodiment, additional targeting groups, in the form ofamino acid side chains, are added to the vinylogous cysteine proteaseinhibitors to form the structure, depicted in Formula 1, above.Generally, this is done by deprotecting the R₁₀ group and coupling anadditional N-protected amino acid, as is well own in the art.

Specifically, the cysteine protease inhibitors of the present inventionthat contain vinylogous esters as the EWG can be generated by thesequence of reactions shown in Scheme I below:

a) Cl—NH₂+(CH₃)OCH₃, dicyclohexylcarboiimide, Et₃N/CH₂Cl₂;

b) LiAlH₄/THF;

c) NaH/THF;

d) HCl/dioxane/CH₂Cl₂; and

e) 4-methylmorpholine, isobutyl chloroformate/THF.

A preferred embodiment treats Boc-protected amino acids such ashomophenylalanine, depicted as the example of Scheme 1, withN,O-dimethylhydroxylamine hydrochloride, in the presence oftriethylamine and dicyclohexylcarbodiimide in dichloromethane.

Alternative embodiments treat the Boc-protected amino acids in thepresence of triethylamine and the coupling reagentbenzotriazol-1-yloxytris (dimethylamino)-phosphonium hexafluorophosphate(BOP). Subsequent reduction with lithium aluminum hydride results inBoc-a-amino aldehydes, for example using the method of Fehrentz, J-A.and Castro, B. (Synthesis, (1983), 676-678). The resulting aldehydes arethen treated with the sodium anion of triethyl phosphonoacetate, in themanner of Wadsworth, W. S. and Emmons, W. D. (J. Am. Chem. Soc. (1961),83, 1733, to produce the vinylogous esters of the present invention.These vinylogous esters are then deprotected with hydrogen chloride indioxane, and coupled with N-protected amino acids to form pseudopeptidylvinylogous esters, the cysteine protease inhibitors of this embodiment.Coupling is acheived via mixed anhydride or other peptide couplingreaction sequences known to those skilled in the art.

In a preferred embodiment, the cysteine protease inhibitors of thepresent invention that contain vinylogous sulfones as the EWG can begenerated by the sequence of reactions shown in Scheme II below:

Generally, the preferred embodiment utilizes Boc-a-amino aldehydes,prepared according to the method of Fehrentz, J-A. and Castro, B.(Synthesis, (1983). Treatment of these aldehydes with the sodium anionof sulfonylmethanephosphonates, for example diethylphenylsulfonylmethanephosphonate, results in the correspondingvinylogous sulfone derivatives, in the manner of Wadsworth, W. S. andEmmons, W. D. (J. Am. Chem. Soc. (1961), 83, 1733. In one embodiment,sulfonyl methanephosphonates are synthesized by coupling of the alkalimetal anion of sulfones such as methyl phenyl sulfone with diethylchlorophosphate.

Alternative embodiments utilize m-chloroperbenzoic acid oxidation ofcommercially available sulfides such as diethylphosphonomethyl methylsulfide (Aldrich Chemical Co.). The vinylogous sulfones are thendeprotected with hydrogen chloride in dioxane, and are coupled withN-protected amino acids to form pseudopeptidyl vinylogous sulfones,using techniques well known in the art.

The vinylogous carboxylates of this embodiment are made using thesequence of reactions shown in Scheme III below:

Generally, saponification of the ester functionality of the vinylogousesters, described above, results in carboxylates. Acidification, as isknown in the art, gives the corresponding carboxylic acids.

The cysteine protease inhibitors of the present invention that containvinylogous phosphonates as the EWG may be synthesized by the sequence ofreactions shown in Scheme IV:

a) NaH/THF;

b) HCl/dioxane; and

c) Mu-PheOH, 4-methylmorpholine, isobutyl chloroformate/THF.

Generally, vinylogous phosphonate derivatives are made by treatingBoc-a-amino aldehydes, prepared according to the method of Fehrentz,J-A. and Castro, B. (Synthesis, (1983), with the sodium anion ofmethylenediphosphonates, for example tetraethyl methylenediphosphonate.The vinylogous phosphonate derivatives are then deprotected withhydrogen chloride in dioxane, and coupled with N-protected amino acidsto form pseudopeptidyl vinylogous phosphonates using techniques known tothose skilled in the art.

The vinylogous amides and peptide derivatives of this invention can begenerated by the sequence of reactions shown in Scheme V.

a) (CH₃)₃SiCH₂CH₂OH, dicyclohexylcarbodiimide (DCC), (C₂H₅)₃N,4-dimethylamino-pyridine, CH₂Cl₂;

b) HCl/dioxane;

c) NaOH/C₂H₅OH;

d) PheOSET, DCC, (C₂H₅)₃N, CH₂Cl₂;

e) NaH, EPAc-PheOSET, THF;

f) Mu-PheOH, 4-methylmorpholine, isobutyl chloroformate, THF; and

g) (n-C₄H₉)₄N+F-, 3 Å molecular sieves, THF.

The N-Boc amino acid, protected as its silylethyl (SET) ester, isN-deprotected, then coupled with the saponification product of triethylphosphonoacetate. The diethyl phosphonoacetyl amino acid derivative isthen coupled, in Wadsworth-Emmons fashion, with the appropriateBoc-protected a-amino aldehyde. Further elongation of the peptidesequence is concluded with fluoride-assisted deprotection of the SETmoiety to afford the vinylogous amide.

Generally, to ensure that the double bond remains intact during thepreparation of the vinylogous amides bound to peptide chains in that theC-terminus is to be converted to the free carboxylate or carboxylic acidgroup, the fluoride-cleavable silyethyl (SET) esters are used. Thussaponification conditions, which cause product mixtures resulting fromboth ester hydrolysis and 1,4-addition of hydroxide to the vinylogousamide at high pH, are avoided. The SET protection scheme thereforepermits clean, smooth preparation of peptide sequences prior to the keyWadsworth-Emmons coupling with an eye to carboxylate formation if theC-terminus is to be a free acid.

The cysteine protease inhibitors of the present invention that containvinylogous ketones, nitriles, sulfoxides, sulfonamides, sulfinamides,sulfonates and sulfoximines as the EWG may be synthesized as followsusing the general scheme outlined in Scheme VI:

wherein

a) Cl—H₂N+(Me)OMe, dicyclohexylcarboniimide, triethylamine; and

b) lithium aluminum hydride.

Structure (I-VIII) are as follows. For the synthesis of cysteineprotease inhibitors with vinylogous ketones as the EWG, structure I isused:

Synthesis of α,β-unsaturated ketones is performed by means of theWadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate. Generally, the aldehyde portion is synthesizedas outlined above. The phosphonate is synthesized by treatment of theenolate anion of methyl or substituted methyl ketones, such as acetoneor acetophenone, with diethyl chlorophosphonate. The enolate anion isgenerated, for example, by treatment of a tetrahydrofuran solution ofdiisopropylamine with butyllithium, followed by addition of the ketoneto the lithium diisopropylamide (LDA) solution (H.O. House, ModernSynthetic Reactions, 2and Ed. (W. Benjamin, Inc., Menlo Park, Calif.,Chapter 9). Following formation of the enolate, diethylchlorophosphonate is added. The Wadsworth-Emmons reagent forms as aconsequence of coupling of the enolate with diethyl chlorophosphate.

For the synthesis of cysteine protease inhibitors with vinylogousnitrites as the EWG, structure II is used:

Synthesis of α,β-unsaturated nitrites is performed by means of theWadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate. Generally, the aldehyde portion is synthesizedas outlined above. The phosphonate is commercially available.

For the synthesis of cysteine protease inhibitors with vinylogoussulfoxides as the EWG, structure III is used:

Synthesis of α,β-unsaturated sulfoxides is performed by means of theWadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate. Generally, the aldehyde portion is synthesizedas outlined above. The phosphonate is synthesized by treatment of theanion of methyl sulfoxides with diethyl chlorophosphate. The anion isgenerated by addition of BuLi to diisopropylamine, followed by additionof the methyl sulfoxide.

For the synthesis of cysteine protease inhibitors with vinylogoussulfonamides as the EWG, structure IV is used:

Synthesis of α,β-unsaturated sulfonamides is performed by means of theWadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate. Generally, the aldehyde portion is synthesizedas outlined above. The phosphonate is synthesized, for instances, by amethod such as the following: a) diethylphosphoryl methanesulfonates, asprepared by the method of Carretero and Ghosez (Tetrahedron Lett.,28:1104-1108 (1987)), are converted to sulfonyl chlorides by treatmentwith phosphorus pentachloride (M. Quaedvlieg, in “Methoden derOrganische Chemic (Houben-Weyl)”, ed. E. Muller, Thieme Verlag,Stuttgart, 4th Ed., 1955, Vol. IX, Chapter 14); or b) treatment of thesulfonyl chloride with an amine, such as ammonia, a primary amine(including an amino acid derivative), or a secondary amine, that resultsin the formation of the sulfonamide (Quaedvlieg, supra, Chapter 19). Thesulfonamide-phosphonate is then reacted with Boc-α-aminoaldehydes toform the target compounds as per the Wadsworth-Emmons reaction.

For the synthesis of cysteine protease inhibitors with vinylogoussulfinamides as the EWG, structure V is used:

Synthesis of α,β-unsaturated sulfinamides is performed by means of theWadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate. Generally, the aldehyde portion is synthesizedas outlined above. The phosphonate may be synthesized using one of thefollowing methods. Treatment of methyl dialkyl phosphonates such as thecommercially available methyl diethyl phosphonate (Aldrich), withthionyl chloride in the presence of aluminum chloride gives thedialkylphosphoryl methanesulfinyl chloride (Vennstra et al., Synthesis(1975) 519. See also Anderson, “Comprehensive Organic Chemistry(Pergamon Press)”, Vol. 3, Chapter 11.18, (1979). Alternatively,treatment of the dialkyl phosphoryl sulfinyl chloride amines (Stirling,Internat. J. Sulfur Chem. (B) 6:277 (1971)), yields the dialkylphosphoryl sulfinamide.

For the synthesis of cysteine protease inhibitors with vinylogoussulfoximines as the EWG, structure VI is used:

Synthesis of α,β-unsaturated sulfoximines is performed by means of theWadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate. Generally, the aldehyde portion is synthesizedas outlined above. The phosphonate may be synthesized in several ways.For example, N-alkyl or N-aryl phenyl methyl sulfoximines are made bythe methods described by Johnson, in “Comprehensive Organic Chemistry(Pergamon Press), supra, Chapter 11.11. Alternatively, the lithium anionof compounds such as N-alkyl phenyl methyl sulfoximine is prepared bythe treatment of the neutral compound with buthyl lithium in THF (Cramet al., J. Amer. Chem. Soc. 92:7369 (1970)). Reaction of this lithiumanion with dialkyl chlorophosphates such as the commercially availablediethyl chlorophosphate (Aldrich) results in the Wadsworth-Emmonsreagent necessary for synthesis of the sulfoximine compounds.

For the synthesis of cysteine protease inhibitors with vinylogoussulfonates as the EWG, structure VII is used:

Synthesis of α,β-unsaturated sulfonates is performed by means of theWadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate, for instance diethylphosphorylmethanesulfonate. The phosphonate may be synthesized as follows. Theanion of methyl dialkyl phosphonates such as the commercially availablemethyl diethyl phosphonate (Aldrich) is generated by treatment of saidphosphonate with a strong base such as LDA. The resulting anion issulfonated with sulfur trioxide/trimethylamine complex (Carreto et al.,Tetrahedron Lett., 28:1104-1108 (1987)) to form diethylphosphorylmethanesulfonate, which is capable of reacting in the Wadsworth-Emmonsprocedure with aldehydes to form α,β-unsaturated sulfonates.

For the synthesis of cysteine protease inhibitors with vinylogous nitrocompoundss as the EWG, structure VIII is used:

Synthesis of α,β-unsaturated nitro compounds is performed by means ofthe Wadsworth-Emmons reaction between Boc-α-amino aldehydes and theappropriate phosphonate. The phosphonate is synthesized by treatment ofthe enolate anion of nitromethane with diethyl chlorophosphate. Theenolate anion is generated, for example, by treatment of atetrahydrofuran solution of diisopropylamine with butyllithium, followedby addition of nitromethane to the lithium diisopropylamide (LDA)solution (House, supra). Following formation of the enolate, diethylchlorophosphate is added. The Wadsworth-Emmons reagent forms as aconsequence of coupling of the nitromethane anion with diethylchlorophosphate.

For the synthesis of cysteine protease inhibitors with EWGs comprisingfive or six homo- or heterocyclic aromatic rings with at least onesubstituted EWG group, scheme VII is used:

Wherein

a) heat at reflux;

b) solvent;

c) H₂O/NaOH;

d) organic extraction;

e) mix;

f) HCl/dioxane 4M;

g) couple;

h) base; and

R₈=the five or six homo- or heterocyclic aromatic rings with at leastone substituted EWM, MDG, or DG; and

R₉=a suitable group as defined above.

The chloride compounds containing R₈ and R₉ groups are generally madeusing commercially available reagents and products using techniques wellknown in the art. The reaction generally produces a mixture of cis andtrans configurations.

Cysteine protease inhibitors containing a diene bond instead of a singlealkene bond are made using the same general synthetic scheme as outlinedabove, by repeating the reaction, as shown below in Scheme VIII:

The cysteine protease inhibitors which contain two EWGs linked to one ofthe carbons of the alkene bond may be synthesized using, for example,the methods disclosed in Brillon et al., J. Org. Chem. 57:1838-1842(1992), hereby expressly incorporated by reference.

In one embodiment, the cysteine protease inhibitors of the presentinvention are further purified if necessary after synthesis, for exampleto remove unreacted materials. For example, the cysteine proteaseinhibitors of the present invention may be crystallized, or passedthrough silica chromatography columns using solvent mixtures to elutethe pure inhibitors.

In particular, the cysteine protease inhibitors of the present inventionsynthesized through the use of the Wittig reaction may be furtherpurified to isolate either the cis or trans configuration. For example,the techniques outlined above may be used.

Once produced, the cysteine protease inhibitors of the present inventionmay be easily screened for their inhibitory effect. The inhibitor isfirst tested against the cysteine protease for that the targeting groupof the inhibitor was chosen, as outlined above. Alternatively, manycysteine proteases and their corresponding chromogenic substrates arecommercially available. Thus, a variety of cysteine proteases areroutinely assayed with synthetic chromogenic substrates in the presenceand absence of the cysteine protease inhibitor, to confirm theinhibitory action of the compound, using techniques well known in theart. The effective inhibitors are then subjected to kinetic analysis tocalculate the k_(irr) and K_(I) values, and the second order rateconstants determined. This is done using techniques well known in theart; for example, progress curve analysis using non-linear curve fittingsoftware can be done to determine the first order rate constants. Suchsoftware is sold under such tradenames as Sigma Plot (Jandel ScientificCorp.) and Statview (Abacus Concepts, Inc.).

If a compound inhibits at least one cysteine protease, it is a cysteineprotease inhibitor for the purposes of the invention. Preferredembodiments have inhibitors that exhibit the correct kinetic parametersagainst at least the targeted cysteine protease.

In some cases, the cysteine protease is not commercially available in apurified form. The cysteine protease inhibitors of the present inventionmay also be assayed for efficacy using biological assays. For example,the inhibitors may be added to cells or tissues that contain cysteineproteases, and the biological effects measured.

In one embodiment, the cysteine protease inhibitors of the presentinvention are synthesized or modified such that the in vivo and in vitroproteolytic degradation of the inhibitors is reduced or prevented.Generally, this is done through the incorporation of synthetic aminoacids, derivatives, or substituents into the cysteine proteaseinhibitor. Preferably, only one non-naturally occurring amino acid oramino acid sidechain is incorporated into the cysteine proteaseinhibitor, such that the targeting of the inhibitor to the enzyme is notsignificantly affected. However, some embodiments that use longercysteine protease inhibitors containing a number of targeting residuesmay tolerate more than one synthetic derivative. In addition,non-naturally occurring amino acid substituents may be designed to mimicthe binding of the naturally occurring side chain to the enzyme, suchthat more than one synthetic substituent is tolerated.

In this embodiment, the resistance of the modified cysteine proteaseinhibitors may be tested against a variety of known commerciallyavailable proteases in vitro to determine their proteolytic stability.Promising candidates may then be routinely screened in animal models,for example using labelled inhibitors, to determine the in vivostability and efficacy.

In one embodiment, the cysteine protease inhibitors of the presentinvention are labelled. By a “labelled cysteine protease inhibitor”herein is meant a cysteine protease inhibitor that has at least oneelement, isotope or chemical compound attached to enable the detectionof the cysteine protease inhibitor or the cysteine protease inhibitorirreversible bound to a cysteine protease. In general, labels fall intothree classes: a) isotopic labels, which may be radioactive or heavyisotopes; b) immune labels, which may be antibodies or antigens; and c)colored or fluorescent dyes. The labels may be incorporated into thecysteine protease inhibitor at any position. For example, a label may beattached as the R₁₀ group in Formula 1. Examples of useful labelsinclude ¹⁴C, ³H, biotin, and fluorescent labels as are well known in theart.

In one embodiment, compositions comprising chiral vinylogousphosphonates are provided. These chiral vinylogous phosphonates arederived from α-amino acids; that is, α-amino acids are used to generatethe (α-amino aldehydes used in the synthesis. This novel class ofcompounds may be used in a preferred embodiment as cysteine proteaseinhibitors.

In one embodiment, compositions comprising chirally pure vinylogoussulfones derived from ax-amino acids are provided. This novel class ofcompounds may be used in a preferred embodiment as cysteine proteaseinhibitors.

Also provided are methods for inhibiting cysteine proteases using thecysteine protease inhibitors of the present invention. In the preferredembodiment, a cysteine protease inhibitor is irreversibly bound to acysteine protease. This is accomplished using ordinary techniques, andwill normally require contacting or adding the cysteine proteaseinhibitor to the sample of the cysteine protease to be inhibited. Thecysteine protease inhibitors of the present invention are stoichiometricinhibitors; that is, a single inhibitor molecule will inhibit a singleenzyme molecule. Thus in the preferred embodiment, an excess ofinhibitor is added to ensure it all or most of the protease isinhibited. In alternative embodiments, only a portion of the proteaseactivity is inhibited. In some embodiments, the cysteine proteaseinhibitor is labelled, such that the presence of a cysteine protease isdetected after the excess inhibitor is removed. This may be done forexample in a diagnostic assay for the detection or quantification ofcysteine proteases in a sample, for example, in blood, lymph, saliva,skin or other tissue samples, in addition to bacterial, fungal, plant,yeast, viral or mammalian cell cultures.

In the preferred embodiment, the cysteine protease inhibitors of thepresent invention are administered to a patient to treat cysteineprotease-associated disorders. By “cysteine protease-associateddisorders” or grammatical equivalents herein is meant pathologicalconditions associated with cysteine proteases. In some disorders, thecondition is associated with increased levels of cysteine proteases; forexample, arthritis, muscular dystrophy, inflammation, tumor invasion,and glomerulonephritis are all associated with increased levels ofcysteine proteases. In other disorders or diseases, the condition isassociated with the appearance of an extracellular cysteine proteaseactivity that is not present in normal tissue. In other embodiments, acysteine protease is associated with the ability of a pathogen, such asa virus, to infect or replicate in the host organism.

Specific examples of cysteine protease associated disorders orconditions include, but are not limited to, arthritis, musculardystrophy, inflammation, rumor invasion, glomerulonephritis, malaria,Aizheimer's disease, disorders associated with autoimmune systembreakdowns, periodontal disease, cancer metastasis, trauma,inflammation, gingivitis, leishmaniasis, filariasis, and other bacterialand parasite-borne infections, and others outlined above.

In particular, disorders associated with interleukin 1β convertingenzyme (ICE) are included, as outlined above.

In this embodiment, a therapeutically effective dose of a cysteineprotease inhibitor is administered to a patient. By “therapeuticallyeffective dose” herein is meant a dose that produces the effects forthat it is administered. The exact dose will depend on the disorder tobe treated and the amount of cysteine protease to be inhibited, and willbe ascertainable by one skilled in the art using known techniques. Ingeneral, the cysteine protease inhibitors of the present invention areadministered at about 1 to about 1000 mg per day. For example, asoutlined above, some disorders are associated with increased levels ofcysteine proteases. Due to the 1:1 stoichiometry of the inhibitionreaction, the dose to be administered will be directly related to theamount of the excess cysteine protease. In addition, as is known in theart, adjustments for inhibitor degradation, systemic versus localizeddelivery, and rate of new protease synthesis, as well as the age, bodyweight, general health, sex, diet, time of administration, druginteraction and the severity of the disease may be necessary, and willbe ascertainable with routine experimentation by those skilled in theart.

A “patient” for the purposes of the present invention includes bothhumans and other animals and organisms. Thus the methods are applicableto both human therapy and veterinary applications. For example, theveterinary applications include, but are not limited to, canine, bovine,feline, porcine, equine, and ovine animals, as well as otherdomesticated animals including reptiles, such as iguanas, turtles andsnakes, birds such as finches and members of the parrot family, rabbits,rodents such as rats, mice, guinea pigs and hamsters, amphibians, andfish. Valuable non-domesticated animals, such as zoo animals, may alsobe treated. In the preferred embodiment the patient is a mammal, and inthe most preferred embodiment the patient is human.

The administration of the cysteine protease inhibitors of the presentinvention can be done in a variety of ways, including, but not limitedto, orally, subcutaneously, intravenously, intranasally, transdermally,intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally,or intraocularly. In some instances, for example, in the treatment ofwounds and inflammation, the cysteine protease inhibitors may bedirectly applied as a solution or spray.

The pharmaceutical compositions of the present invention comprise acysteine protease inhibitor in a form suitable for administration to apatient. In the preferred embodiment, the pharmaceutical compositionsare in a water soluble form, such as being present as pharmaceuticallyacceptable salts, which is meant to include both acid and base additionsalts. “Pharmaceutically acceptable acid addition salt” refers to thosesalts that retain the biological effectiveness of the free bases andthat are not biologically or otherwise undesirable, formed withinorganic acids such as hydrochloric acid, hydrobromic acid, sulfuricacid, nitric acid, phosphoric acid and the like, and organic acids suchas acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalicacid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaricacid, citric acid, benzoic acid, cinnamic acid, mandelic acid,methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid,salicylic acid and the like. “Pharmaceutically acceptable base additionsalts” include those derived from inorganic bases such as sodium,potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,manganese, aluminum salts and the like. Particularly preferred are theammonium, potassium, sodium, calcium, and magnesium salts. Salts derivedfrom pharmaceutically acceptable organic non-toxic bases include saltsof primary, secondary, and tertiary amines, substituted amines includingnaturally occurring substituted amines, cyclic amines and basic ionexchange resins, such as isopropylamine, trimethylamine, diethylamine,triethylamine, tripropylamine, and ethanolamine.

The pharmaceutical compositions may also include one or more of thefollowing: carrier proteins such as serum albumin; buffers; fillers suchas microcrystalline cellulose, lactose, corn and other starches; bindingagents; sweeteners and other flavoring agents; coloring agents; andpolyethylene glycol. Additives are well known in the art, and are usedin a variety of formulations.

The following examples serve to more fully describe the manner of usingthe above-described invention, as well as to set forth the best modescontemplated for carrying out various aspects of the invention. It isunderstood that these examples in no way serve to limit the true scopeof this invention, but rather are presented for illustrative purposes.All references cited herein are expressly incorporated by reference.

EXAMPLES

The following abbreviation conventions have been used to simplify theexamples.

Mu=morpholine urea

Xaa₁=amino acid at P1 position relative to active site of the enzyme

Xaa₂=amino acid at P2 position relative to active site of the enzyme

Xaa_(1′)=amino acid at P1′ position relative to active site of theenzyme

VSMe=vinyl sulfone with methyl terminus

VA=vinyl carboxylate

VAM-Xaa_(1′)=vinyl amide coupled to amino acid 3;

VEOEt=vinyl ethyl ester

VPEt=vinyl phosphonate

VSPh=vinyl sulfone with phenyl terminus

Hph=homophenylalanine

For instance, Mu-Phe-Lys(Z)VSPh, where Xaa₂=Phe (phenylalanine) andXaa₁=Lys(Z) (lysine, protected as its carbobenzyloxy urethane),transformed to the phenyl vinyl sulfone according to the proceduredescribed in Example 5 and depicted in Scheme 2.

Example 1 Synthesis of Cysteine Protease Inhibitor Containing aVinylogous Ethyl Ester

Unless otherwise indicated, all reactions were performed under an inertatmosphere of argon or nitrogen at room temperature. THF was distilledfrom sodium benzophenone ketyl. All other solvents and commerciallyavailable reagents were used without further purification.

Synthesis of Ethyl(S)-(E)-4-(4-morpholinecarbonyl-phenylalanyl)amino-6-phenyl-2-hexenoate,abbreviated Mu-Phe-HphVEOEt, was as follows. Unless otherwise noted, allreagents were obtained from Aldrich, Inc. 0.393 g of a 60% mineral oildispersion (9.82 mmol) of sodium hydride was added to a solution oftriethyl phosphonoacetate (2.20 g, 9.82 mmol) in THF (50 mL) at −10° C.The mixture was stirred for 15 minutes, whereupon solution ofBoc-homophenylalaninal (Boc-HphH) (2.35 g, 9.82 mmol, prepared byconversion of Boc-homophenylalanine (Synthetech) to itsN,O-dimethylhydroxamide, using the Fehrentz method, followed by lithiumaluminum hydride reduction) in THF (20 mL) was added. The mixture wasstirred for 45 minutes. 1M HCl (30 mL) was added. The product wasextracted with ethyl acetate (50 mL), washed with saturated aqueousNaHCO₃ (30 mL) dried over MgSO₄, filtered, and evaporated to dryness.The dried material was dissolved in CH₂Cl₂ (10 mL), and a 4.0 M solutionof HCl in dioxane (20 mL) was added. The mixture was stirred for 30minutes. The solvents were removed under reduced pressure and theresidue, ethyl (S)-4-amino-6-phenyl-2-hexenoate hydrochloride, waspumped dry.

Morpholinecarbonylphenylalanine (2.74 g, 9.82 mmol, prepared accordingto the method described in Esser, R. et.al., Arthritis & Rheumatism(1994), 0000) was dissolved in THF (50 mL) at −10° C. 4-methylmorpholine(1.08 mL, 9.82 mmol) was added, followed by isobutyl chloroformate (1.27mL, 9.82 mmol). The mixed anhydride was stirred for 10 minutes,whereupon a solution of ethyl (S)-4-amino-6-phenyl-2-hexenoatehydrochloride from the previous step in DMF (10 mL) was added, followedby 4-methylmorpholine (1.08 mL, 9.82 mmol). The mixture was stirred for1 hour. 1M HCl (50 mL) was added. The product was extracted with ethylacetate (100 mL), washed with saturated aqueous NaHCO₃ (50 mL), driedover MgSO₄ and decolorizing charcoal (DARCO), filtered, and evaporatedto dryness, giving 3.80 g (80% yield from Boc-homophenylalaninal).

Thin-layer chromatography (TLC) was done. Unless otherwise noted, alldata for this and subsequent examples used 10% MeOH/CH₂Cl₂.Visualization was accomplished by means of UV light at 254 nm, followedby ninhydrin, bromocresol green, or p-anisaldehyde stain. The retentionfactor (Rf) of the Mu-Phe-HphVEOEt was 0.55.

NMR spectra were recorded on a Varian Gemini 300 MHz instrument. All ¹HNMR data of this and subsequent examples are reported as delta values inparts per million relative to internal tetramethylsilane, peakassignments in boldface. The following abbreviations are used: s,singlet; d, doublet; t, triplet; q, quartet; br, broad. An asterisk (*)implies that a signal is obscured or buried under another resonance.

¹H NMR (CDCl₃): 1.28 (3H, t, J=6 Hz, CH₃); 1.7-1.95 (m, 2H, CH₂CH₂Ph);2.59 (2H, CH₂CH₂Ph); 2.99-3.17 (2H, 2×dd, J=7,15 Hz and 5,15 Hz, PhCH₂of Phe residue); 3.26 (4H, m, 2×CH₂O (morpholine)); 3.61 (4H, m, 2×CH₂N(morpholine)); 4.39 (q, J=6 Hz, CH₂ (ester)); 4.47-4.6 (2H, m*, 2×CHNH);5.06 (1H, d, J=7 Hz, NHCH), 5.65 (1H, d, J=15 Hz, trans COCH═CH,); 6.15(1H, d, J=7 Hz, NHCH); 6.67 (1H, dd, J=5,15 Hz, trans CH═CHCO);7.09-7.36 (10H, m, aromatic).

Mass spectral data were obtained from M-Scan, Inc., West Chester, Pa.FAB, high-resolution: calculated for C₂₈H₃₅N₃O₅, (m+H)=494.2655, found,494.2638.

Indication of approximately a 7:1 ratio of (S) to (R) epimers wasevidenced by the appearance of a doublet at 5.82 ppm (J=15 Hz) coupledto a doublet of doublets at 6.79 (J=5,15 Hz). This phenomenon ispossibly due to isomerically impure commercial material.

Example 2 Synthesis of Cysteine Protease Inhibitor Containing aVinylogous Ethyl Ester

Ethyl(S)-(E)-7-guanidino-4-(4-morpholinecarbonylphenylalanyl)amino-2-heptenoatehydrobromide, abbreviated MU-Phe-ArgVEOEt.HBr, was made as follows.Boc-N_(g)-4methoxy-2,3,6-trimethylbenzenesulfonylarginineBoc-Arg(Mtr)OH) was converted to its aldehyde, abbreviated asBoc-Arg(Mtr)H according to the method of Fehrentz and Castro, supra. Toa solution of triethyl phosphonoacetate (0.916 mL, 3.61 mmol) in THF (20mL) was added NaH (0.145 g of a 60% mineral oil dispersion, 3.61 mmol).The solution was stirred for 15 minutes. A solution of Boc-Arg(Mtr)H(1.5 g, 3.29 mmol) in THF (5 mL) was added. The mixture was stirred for45 minutes. 1HCl (30 mL) was added. The product was extracted into ethylacetate (50 mL), washed with saturated aqueous NaHCO₃ (30 mL), driedover MgSO₄, filtered, and evaporated to dryness. The residue wasprecipitated from CH₂Cl₂/ether/hexane to give 1.55 g (89%) of product,ethyl(S)-(E)-4-tert-butoxycarbonylamino-7-(4methoxy-2,3,6trimethylbenzenesulfonylguanidino)-2-heptenoate.

This material was treated immediately with 5 mL of 30% hydrogen bromidein acetic acid for 5 hours. The orange oil was dissolved in methanol (8mL), and poured into ether (300 mL). The oil that separated was dried invacuo, giving 0.81 g (67%) of ethyl (S)-4-amino-7-guanidino-2-heptenoatedihydrobromide (ArgVEOEt.2HBr) as an orange foamy solid.

To a solution of Mu-PheOH (0.55 g, 1.98 mmol) in DMF (8 mL) at −10° C.were added 4-methylmorpholine (0.217 mL, 1.98 mmol), followed byisobutyl chloroformate (0.256 mL, 198 mmol). The mixed anhydride wasstirred for 15 minutes, whereupon a solution of ArgVEOEt.2HBr (0.81 g,1.98 mmol) in DMF (2 mL) was added, followed by 4-methylmorpholine(0.217 mL, 1.98 mmol). The mixture was stirred for 1 hour. The solventwas removed in vacuo. Butanol (30 mL) was added. The solution was washedwith saturated aqueous NaHCO₃ (15 mL), filtered through glass wool, andthe solvent was removed under reduced pressure. The residue wasdissolved in methanol (5 mL) and filtered through a pad of Celite. Thefiltrate was poured into 1:1 ether/ethyl acetate (300 mL), filtered, andthe solids were dried overnight, giving 0.77 g of product (68%: 41% fromBoc-Arg(Mtr)H).

Melting points for this and subsequent examples were recorded on aMel-Temp II. The melting point of the ArgVEOEt.HBr was 138-142° C.(dec.).

The NMR results, as defined in Example 1, were as follows. NMR(DMSO-d⁶): 1.2 (3H, t, J=6 Hz, CH₃); 1.4-1.65 (4H, m,CH₂CH₂CH₂-guanidinium); 2.82-2.95 (2H, m, CH₂Ph); 3.0-3.15 (2H, m*,CH₂-guanidinium); 3.1 (4H, m, 2×CH₂N (morpholine)); 3.45 (4H, m, 2×CH₂O(morpholine)); 4.1 (2H, q, J=6 Hz, CH₂ (ester)); 4.18 (1H, m, CHNH);4.43 (1H, m, CHNH); 5.75 (1H, d, J=15 Hz, trans COCH═CH); 6.67 (1H, d,J=7 Hz, NHCH); 6.75 (1H, dd, J=5,15 Hz, trans CH═CHCO); 7.1-7.3 (9H, m*,aromatic and guanidinium); 7.81 (1H, m, NH); 8.15 (1H, d, J=7 Hz, NHCH).

Example 3 Additional Cysteine Protease Inhibitors with Vinylogous EthylEsters

(S)-(E)-Ethyl8-(benzyloxycarbonyl)amino-4-(4-morpholinecarbonylphenylalanyl)amino-2-octenoateand (S)-(E)-Ethyl8-amino-4-(4-morpholinecarbonylphenylalanyl)amino-2-octenoatehydrobromide were made as per Examples 1 and 2, and tested against avariety of cysteine proteases as shown below.

Example 4 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone with Methyl terminus

Synthesis of(S)-(E)-5-(4-morpholinecarbonyl-phenylalanyl)amino-7-phenyl-2-thia-3-heptene2,2-dioxide, abbreviated Mu-Phe-HphVSMe, was as follows.Diethyl(methylsulfonylmethyl) phosphonate (MSMP) was prepared bym-chloroperbenzoic acid oxidation of commercially availablediethyl(methylthiomethyl) phosphonate. To a solution of MSMP (1.5 g,6.51 mmol) in THF (25 mL) at 0° C. was added sodium hydride (0.26 g of a60% mineral oil dispersion). The mixture was stirred for 15 minutes. Asolution of Boc-HphH (1.72 g, 6.51 mmol) in THF (10 mL) was added. Themixture was stirred for 1 hour. 1M HCl (30 mL) was added. The productwas extracted into ethyl acetate (50 mL), washed with saturated aqueousNaHCO₃ (30 mL), dried over MgSO₄, filtered, and the solvents wereremoved under reduced pressure, giving 1.90 g of a white solid.

This material was dissolved in CH₂Cl₂ (5 mL), and was treated with 10 mLof a 4.0 M solution of HCl in dioxane. The mixture was stirred for 2hours. Ether (200 mL) was added to the white suspension. The mixture wasstirred vigorously for 5 minutes, filtered, and pumped dry to give 1.40g of (S)-(E)-5-amino-7-phenyl-2-thia-3-heptene 2,2-dioxide(HCl.HphVSMe).

To a solution of Mu-PheOH (0.71 g, 2.54 mmol) in THF (10 mL) at −10° C.were added 4-methylmorpholine (0.28 mL, 2.54 mmol) and isobutylchloroformate (0.329 mL, 2.54 mmol). The mixed anhydride was stirred for10 minutes, whereupon a solution of HCl.HphVSMe (0.70 g, 2.54 mmol) inDMF (2 mL) was added, followed by 4-methylmorpholine (0.28 mL, 2.54mmol). The mixture was stirred for 45 minutes. 1M HCl (30 mL) was added.The product was extracted into ethyl acetate (50 mL), washed withsaturated aqueous NaHCO3 (30 mL), dried over MgSO4, filtered, andevaporated to dryness. The residue was crystallized fromCH₂Cl₂/ether/hexane (1:10:5) to give a total of 0.57 g (45% yield, 35%from Boc-HphH).

The retention factor on TLC: (10% MeOH/CH2Cl2) was 0.69. ¹H NMR (CDCl3):1.7-1.97 (2H, m, CH₂CH₂Ph); 2.31 (2H, m, CH₂CH₂Ph); 2.86 (3H, s,CH₃SO₂); 3.08 (2H, br. d, PhCH₂); 3.48 (4H, m, 2×CH₂N (morpholine));3.62 (4H, m, 2×CH₂O, (morpholine)); 4.52 (1H, q, J=7 Hz, CHNH (Phe));4.61 (1H, m, C_(H)NH); 4.95 (1H, d, J=7 Hz, NHCH); 6.10 (1H, dd, J=2,15Hz, trans SO₂CH═CH); 6.43 (1H, d, J=7 Hz, NHCH); 6.70 (1H, dd, J=5,15Hz, trans CH═CHSO₂); 7.08-7.38 (10H, m, aromatic.)

Indication of approximately an 8:1 ratio of (S) to (R) epimers wasevidenced by the presence in the spectrum of Mu-Phe-HphVSMe of a doubletof doublets at 6.95 ppm (J=5,15 Hz), along with a singlet at 2.87 ppm.This phenomenon is possibly due to isomerically impure commercialmaterial.

Example 5 Synthesis of additional Cysteine Protease Inhibitors withVinylogous Sulfones with Methyl Termini

(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-methylsulfonyl-1-butene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-methylsulfonyl-4-phenyl-1-butene,and(S)-(E)-3-(tert-butoxycarbonylalanyl)amino-1-methylsulfonyl-4-phenyl-1-butenewere made as above, and tested against various cysteine proteases asshown below.

Example 6 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone with Phenyl terminus

Synthesis of(S)-(E)-3-tert-butoxycarbonylamino-4-methyl-1-phenylsulfonyl-1-pentene,abbreviated Boc-ValVSPh, was as follows. To a solution of diethylphenylsulfonylmethylenephosphonate (PSMP), (1.40 g, 4.79 mmol, preparedby treatment of the lithium anion of methyl phenyl sulfone with diethylchlorophosphate) in THF (20 mL) at −20° C. was added sodium hydride(0.192 g of a 60% mineral oil dispersion, 4.79 mmol). The mixture waswarmed to 0° C. over 20 minutes. Boc-valinal (0.876 g, 4.35 mmol)prepared according to the method of Fehrentz and Castro, supra), wasadded as a solution in THF (5 mL). The mixture was stirred for 30minutes. 1M HCl (30 mL) was added. The product was extracted with ethylacetate (50 mL), washed with saturated aqueous NaHCO₃ (30 mL), brine (20mL), dried over MgSO₄, filtered, and evaporated to dryness to afford theproduct in quantitative yield.

The melting point of the Boc-ValVSPh was 101-103° C.

TLC analysis (30% ethyl acetate/pet. ether) showed a retention factor of0.4.

¹H NMR (CDCl₃): 0.92 (6H, 2×d, J=6 Hz, isopropyl CH₃'s of valineresidue); 1.39 (9H, s, t-C₄H₉); 1.87 (1H, m, CH(CH₃)₂); 4.11 (1H, m,CHNH); 4.5 (1H, br d, NHCH); 6.41 (1H, d, J=15 Hz, trans SO₂CH═CH); 6.87(1H, dd, J=5,15 Hz, trans C_(H)═CHSO₂); 7.48-7.87 (5H, m, aromatic).

Example 7 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone with Phenyl Terminus

Synthesis of(S)-(E)-3-(4-morpholinecarbonyl-phenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,abbreviated Mu-Phe-HphVSPh, was as follows. To a solution of PSMP (1.94g, 6.64 mmol) in THF (30 mL) at 0° C. was added sodium hydride (0.27 gof a 60% mineral oil dispersion, 6.64 mmol). After 15 minutes, asolution of Boc-HphH (1.59 g, 8.94 mmol) in THF (5 mL) was added. Themixture was stirred for 30 minutes. 1M HCl (20 mL) was added. Theproduct (Boc-HphVSPh) was extracted into ethyl acetate (80 mL), washedwith saturated aqueous NaHCO₃ (30 mL), dried over MgSO₄, filtered, andthe solvents were removed under reduced pressure to give a pale yellowoil.

This material was dissolved in CH₂Cl₂ (5 mL) and treated with a 4.0 Msolution of HCl in dioxane (15 mL) for 30 minutes. The solvents wereremoved under reduced pressure, the residue was dissolved in methanol (5mL) and the solution was poured into hexane/ether (1:1; 350 mL). Theprecipitate was filtered and pumped dry to give 0.77 g (38% fromBoc-HphH) of (S)-(E)-3-amino-5-phenyl-1-phenylsulfonyl-1-pentenehydrochloride (HCl.HphVSPh).

To a solution of Mu-PheOH (0.63 g, 2.28 mmol) in THF (10 mL) at −10° C.was added 4-methylmorpholine (0.251 mL, 2.28 mmol), followed by isobutylchloroformate (0.295 mL, 2.28 mmol). The mixed anhydride was stirred for10 minutes, whereupon a solution of HCl.HphVSPh (0.77 g, 2.28 mmol) inDMF (2 mL) was added, followed by 4-methylmorpholine (0.251 mL, 2.28mmol). The mixture was stirred for 1 hour. 1M HCl (20 mL) was added. Theproduct was extracted into ethyl acetate (50 mL), washed with saturatedaqueous NaHCO₃ (30 mL), dried over MgSO₄, filtered, and evaporated todryness, giving 0.80 g (62%) yield of product as an amorphous solid.

The melting point of the product was 85-87° C.

¹H NMR (CDCl₃): 1.68-1.93 (2H, m, CH₂CH₂Ph); 2.58 (2H, m, CH₂CH₂Ph);3.03 (2H, d, J=7 Hz, PhCH₂CH); 3.27 (4H, m, 2×CH₂N (morpholine)); 3.62(4H, m, 2×CH₂O (morpholine)); 4.44 (1H, q, J=7 Hz, CHCH₂Ph); 4.62 (1H,m, CHCH═CH); 4.93 (1H, d, J=7 Hz, NHCH); 6.08 (1H, dd, J=2,15 Hz, transSO₂CH═CH); 6.23 (1H, d, J=7 Hz, NHCH); 6.77 (1H, dd, J=5,15 Hz, transCH═CHSO₂); 7.07-7.87 (15H, m, aromatic). Mass Spectroscopy (FAB, highresolution): calculated for C₃₁H₃₅N₃O₅S, (m+H)=562.2376, found 562.2362.

Example 8 Synthesis of Additional Cysteine Protease Inhibitors withVinylogous Sulfones with Phenyl Termini

The following cysteine protease inhibitors were made as per examples 6and 7 and tested as shown below:(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-6-guanidino-1-phenylsulfonyl-1-hexene hydrobromide,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S )-(E)-3-glycylamino-4-phenyl-1-phenylsulfonyl-1-butene hydrochloride,(S)-(E)-7-(benzyloxycarbonyl)amino-3-(4-morpholinecarbonylphenylalanyl)-amino-1-phenylsulfonyl-1-heptene,(S)-(E)-7-amino-3-(4-morpholinecarbonylphenylalanyl)amino-1-phenylsulfonyl-1-heptenehydrobromide,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-4-methyl-1-phenylsulfonyl-1-butene,(S)-(E)-3-amino-4-phenyl-1-phenylsulfonyl-1-butene hydrochloride,(S)-(E)-3-(4-morpholinecarbonylvalyl)amino-4-phenyl-1-phenylsulfonyl-1-butene,(S)-(E)-3-(4-morpholinecarbonylarginyl)amino-6-guanidino-1-phenylsulfonyl-1-hexenedihydrobromide,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-4-benzyloxy-1-phenylsulfonyl-1-butene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-7-benzoylamino-1-phenylsulfonyl-1-heptene,(R)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-[4-morpholinecarbonyl-(3,5-diiodotyrosyl)]-amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-[4-tert-butoxycarbonyl-(3,5-diiodotyrosyl)]-amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S,S)-(E)-3-[4-morpholinecarbonyl-(1,2,3,4-tetrahydro-3-isoquinoline-carbonyl)]amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S,S)-(E)-3-[tert-butoxycarbonyl-(1,2,3,4-tetrahydro-3-isoquinolinecarbonyl)]-amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonylleucylleucyl)amino-4-(4-hydroxyphenyl)-1-phenylsulfonyl-1-butene,(S)-(E)-3-amino-5-phenyl-1-phenylsulfonyl-1-pentene hydrochloride,(S)-(E)-3-[(4-morpholinecarbonyl-(R,S)-α-methylphenylalanyl]amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(benzyloxycarbonylleucylleucyl)amino-4-(4-hydroxyphenyl)-1-phenylsulfonyl-1-butene,(S)-(E)-3-(4-morpholinecarbonyltyrosyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(tert-butoxycarbonyl-2-naphthylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonyl-2-naphthylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonyl-4-biphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-phenylsulfonyl-1-heptene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-phenylsulfonyl-6-thia-1-heptene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-5-methylsulfonyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(acetylleucylleucyl)amino-1-phenylsulfonyl-1-heptene,(S)-(E)-3-(acetylleucylleucyl)amino-1-phenylsulfonyl-6-thia-1-heptene,(S)-(E)-3-(acetylleucylleucyl)amino-5-methylsulfonyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(carbomethoxypropionylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-7-(benzyloxycarbonyl)amino-3-(4-morpholinecarbonylphenylalanyl)-amino-1-fluoro-1-phenylsulfonyl-1-heptene,(S)-(E)-3-(acetylleucylleucyl)amino-4-(4-hydroxyphenyl)-1-phenylsulfonyl-1-butene,(S)-(E)-3-(dimethylsulfamoylphenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-(4-bromophenylsulfonyl)-5-phenyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-(2-napthylsulfonyl)-5-phenyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonyl-2-naphthylalanyl)amino-1-(2-napthylsulfonyl)-5-phenyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-methylsulfonyl-1-butene,(S)-(E)-3-(4-morpholinecarbonylphenylalanyl)amino-1-methylsulfonyl-4-phenyl-1-butene,and(S)-(E)-3-(tert-2butoxycarbonylalanyl)amino-1-methylsulfonyl-4-phenyl-1-butene.

Example 9 Synthesis of a Cysteine Protease Inhibitor with a VinylogousCarboxlate

Synthesis of(S)-(E)-4-(4-morpholinecarbonyl-phenylalanyl)amino-6-phenyl-2-hexenoicacid, abbreviated Mu-Phe-HphVA, was as follows. To a solution ofMu-Phe-VEOEt (see Example 1; 1.96 g, 4.15 mmol) in ethanol (7 mL) wasadded 1M NaOH (9.5 mL) over 3 hours. The solvents were evaporated. Water(30 mL) was added. The solution was washed with ethyl acetate (30 mL),acidified with 1M HCl (15 mL), extracted with ethyl acetate (60 mL),dried over MgSO₄, filtered, and evaporated to dryness, giving a slightlyoff-white foam. Yield=1.63 g (89%).

The melting point was determined to be 91-93° C. TLC analysis in 10%CH₃OH/CH₂Cl₂ showed a retention factor of 0-0.3 (streak, staining yellowwith bromocresol green, indicating presence of acidic functionality).The material was derivatized with HCl.Leu-ProOCH₃ (Example 8, infra, todemonstrate retention of double bond configuration; see NMR datadescribed therein.

Example 10 Synthesis of an Additional Cysteine Protease Inhibitor with aVinylogous Carboxylate

(S)-(E)-Benzyl4-(4-morpholinecarbonylphenylalanyl)amino-6-phenyl-2-hexenamide wassynthesized using the techniques of example 9 and tested as shown below.

Example II Synthesis of a Cysteine Protease Inhibitor with a VinylogousPhosphonate

Synthesis of diethyl(S)-(E)-4-(4-morpholinecarbonylphenylalanyl)amino-6-phenyl-2-hexenephosphonate,abbreviated Mu-Phe-HphVPEt, was as follows. To a solution of tetraethylmethylenediphosphonate (2.00 g, 6.94 mmol) in THF (30 mL) was addedsodium hydride (0.278 g of a 60% mineral oil dispersion, 6.94 mmol). Themixture effervesced rapidly and then clarified. After 5 minutes, asolution of Boc-HphH (1.83 g, 6.94 mmol) in THF (5 mL) was added. Themixture was stirred for 1 hour. 1M HCl (20 mL) was added. The productwas extracted into ethyl acetate (50 mL), washed with saturated aqueousNaHCO₃ (20 mL), brine (10 mL), dried over MgSO₄, filtered, andevaporated to dryness, giving 2.46 g (89%) of diethyl(S)-(E)-4-tert-butoxycarbonylamino-6-phenyl-2-hexenephosphonate(Boc-HphVPEt) as a single spot on TLC (Rf=0.58, 10% CH₃OH/CH₂Cl₂). Thismaterial was used without further purification.

To a solution of Boc-Hph-VPEt (2.46 g, 6.19 mmol) in CH₂Cl₂ (3 mL) wasadded 10 mL of a 4.0 M solution of HCl in dioxane. The mixture wasstirred at room temperature for 1.5 hours. The solvents were removedunder reduced pressure and the residue was dissolved in methanol (10mL). The solution was poured into ether (400 mL). The precipitate wascollected on a Buchner funnel, washed with ether (2×20 mL), and waspumped dry to give 1.25 g (60%) of product, diethyl(S)-(E)-4-amino-6-phenyl-2-hexenephosphonate hydrochloride(HCl.HphVPEt).

To a solution of Mu-PheOH (1.04 g, 3.74 mmol) in THF (15 mL) at −10° C.was added 4-methylmorpholine (0.412 mL. 3.74 mmol), followed by isobutylchloroformate (0.486 mL, 3.74 mmol). The mixed anhydride was stirred for5 minutes, whereupon a solution of HCl.HphVPEt (1.25 g, 3.74 mmol) inDMF (5 mL) was added, followed by 4-methylmorpholine (0.412 mL, 3.74mmol). The mixture was stirred for 1 hour. Ethyl acetate (50 mL) wasadded, The solution was washed with 1M HCl (25 mL), saturated aqueousNaHCO₃ (25 mL), and brine (10 mL), dried over MgSO₄, filtered, andevaporated to dryness. The product, upon treatment withCH₂Cl₂/ether/hexane (315 mL in a 15:200:100 ratio) formed an oil thatsolidified on drying in vacuo to give 1.44 g (69%) of the final product

The melting point of the product was determined to be 53-55° C.

TLC analysis (10% CH₃OH/CH₂Cl₂) showed a retention factor of 0.48.

¹H NMR (CDCl₃): 1.35 (6H, 2×t, J=7 Hz, 2×CH₃CH₁OP); 1.68-1.87 (2H, m,CH₂CH₂Ph); 2.58 (2H, m, CH₂CH₂Ph); 3.08 (2H, d, J=7 Hz, CH₂Ph); 3.28(4H, m, 2×CH₂N (morpholine)); 3.62 (4H, m, 2×CH₂O (morpholine));4.0-4.13 (4H, 2×dq, J=3,7 Hz, 2×CH₃CH₂OP, long-range H—C—O—P couplingresponsible for splitting of quartet); 4.53 (1H, q*, J=7 Hz, CHNHC═O);4.56 (1H, m, CHCH═CH); 5.02 (1H, d, J=7 Hz, NHCH); 5.50 (1H, ddd,J=2,15,17 Hz, P—CH═CH—CH); 6.34 (1H, d, J=7 Hz, NHCH); 6.58 (1H, ddd,5,15,20 Hz, CH═CH—P); 7.08-7.34 (10H, m, aromatic).

Mass spectroscopy (FAB, high resolution): calculated for C₂₉H₄₀N₃O₆P,(m+H)=558.2733, found 558.2775.

Example 12 Synthesis of a Cysteine Protease Inhibitor with A VinylogousAmide

Synthesis of (S)-(E)-(N-leucylproline methylester)-4-(4-morpholinecarbonylphenyl-alanyl)amino-6-phenyl-2-hexenamide,abbreviated Mu-Phe-HphVAM-Leu-ProOMe, was as follows. Leucylprolinemethyl ester hydrochloride (HCl.Leu-Pro-OMe) was prepared by mixedanhydride coupling of Boc-leucine and proline methyl esterhydrochloride, followed by Hcl-mediated deprotection of the N-terminalBoc group. Mu-Phe-HphVA (from Example 6, 1.42 g, 3.21 mmol) wasdissolved in THF (15 mL) and cooled to −10° C. To this solution wasadded 4-methylmorpholine (0.354 mL, 3.21 mmol), followed by isobutylchloroformate (0.417 mL, 3.21 mmol). The mixed anhydride was stirred for5 minutes, whereupon HCl.Leu-ProOMe (0.897 g, 3.21 mmol) was added.4-methylmorpholine (0.354 mL, 3.21 mmol) was added. The mixture wasstirred for 4 hours. 1M HCl (20 mL) was added. The product was extractedinto ethyl acetate (50 mL), washed with saturated aqueous NaHCO₃ (20mL), brina (20 mL, dried over MgSO₄, filtered, and evaporated todryness. The residue was dissolved in CH₂Cl₂ (5 mL) and was poured into1:1 ether/pet ether (150 mL). The precipitate was collected on a Buchnerfunnel and pumped dry to give 1.42 g (64%) of a white solid.

The melting point of the product was determined to be 97-100° C.

TLC analysis (10% CH₃OH/CH₂Cl₂) showed a retention factor of 0.47.

¹H NMR (CDCl₃): 0.93 (6H, 2×d, J=7 Hz, 2×CH₃ (Leu)); 1.67-2.25 (9H, mCH(CH₃)₂, CH₂ (Leu), 2×CH₂ (Pro), CH₂CH₂Ph); 2.54 (1H, br t, PhCH₂CH₂);3.04 (2H, m, CH2Ph); 3.28 (4H, m, 2×CH₂N (morpholine)); 3.5-3.8 (6H, m*,2×CH₂O (morpholine), CH₂N (Pro)); 3.7 (3H, s, OCH₃); 4.42-4.52 (2H, m,2×CHNH); 4.89 (1H, m, CHNH); 5.11 (1H, d, J=7 Hz, NHCH); 5.43 (1H, dd,J=2,15 Hz, trans NHCOCH═CH); 6.03 (1H, d, J=7 Hz, NHCH); 6.25 (1H, d,J=7 Hz, NHCH); 6.55 (1H, dd, J=6,15 Hz, trans CH═CHCONH); 7.03=7.36(10H, m, aromatic).

Example 13 Synthesis of a Cysteine Protease Inhibitor with a VinylogousAmide

Synthesis of(S)-(E)-(N-phenylalanine)-4-(4-morpholinecarbonylphenylalanyl)amino-6-phenyl-2-hexenamide,abbreviated Mu-Phe-HphVAM-PheOH, proceeded as follows. Boc-PheOH (5.00g, 18.85 mmol), trimethylsilylethanol (2.70 mL, 18.85 mmol), and4-dimethylaminopyridine (0.23 g, 1.89 mmol) were dissolved in CH₂Cl₂ (70mL). A solution of dicylcohexylcarbodiimide (DCC) (3.89 g, 18.85 mmol)in CH₂Cl₂ (20 mL) was added. The mixture was stirred for 1 hour. Thesuspension was filtered and the solvent was removed under reducedpressure. The residue was dissolved in ether (200 mL), washed with 50 mLeach of 1M HCl, saturated aqueous NaHCO₃, and brine, dried over MgSO₄,filtered, and evaporated to dryness. The residue was dissolved in hothexane, filtered to remove any residual dicyclohexylurea (DCU)by-product, and evaporated to give 6.7 g (98% yield) of a colorless oil,tert-butoxycarbonylphenylalanine, silylethyl ester (Boc-PheOSET). TLC ofthe Boc-PheOSET (30% ethyl acetate/hexane) showed a Rf of 0.65.

30 mL of a 4.0 M solution of HCl in dioxane was added to the Boc-PheOSETfrom the previous step. The mixture was stirred for 90 minutes. Thesolvents were evaporated, giving a waxy solid, phenylalanine silylethylester hydrochloride (HCl.PheOSET).

¹H NMR (CDCl₃): 0.02 (9H, s, (CH₃)₃Si); 0.89 (2H, m, CH₂Si); 3.32-2.51(2H, 2×dd, J=7,14 Hz, J=5,14 Hz, CH₂Ph); 4.18 (2H, m, CH₂CH₂Si); 4.37(1H, br q, CHNH₃ ⁺); 7.23-7.37 (5H, m, aromatic); 8.82 (3H, br s, NH₃⁺).

Diethyl phosphonoacetic acid (DEPA) was prepared in quantitative yieldby saponification of triethyl phosphonoacetate in ethanol, in thepresence of 1.1 equivalents of 1M NaOH. To a solution of DEPA (1.62 g,8.28 mmol), HCl.PheOSET (2.5 g, 8.28 mmol), and triethylamine (0.676 mL,8.28 mmol) in CH₂Cl₂ (30 mL) was added a solution of DCC (1.71 g, 8.28mmol) in CH₂Cl₂ (10 mL). The mixture was stirred at room temperature for16 hours. The white suspension was filtered, the solvent was removedunder reduced pressure, and the residue was dissolved in ethyl acetate(100 mL). The solution was washed with 1M HCl (50 mL), saturated aqueousNaHCO₃ (50 mL), brine (25 mL), dried over MgSO₄, filtered, andevaporated to dryness. The residue was dissolved in hot pet ether andfiltered to remove any remaining DCU, and was evaporated to give acolorless oil, diethyl phosphonoacetylphenylalanine, silylethyl ester(EPAc-PheOSET) weighing 3.56 g (97% from Boc-PheOSET). TLC analysis (20%ethyl acetate/CH₂Cl₂) showed a retention factor of 0.22.

To a solution of EPAc-PheOSEt (1.19 g, 2.68 mmol) in THF (10 mL) at 0°C. was added sodium hydride (107 mg of a 60% mineral oil dispersion).The mixture was stirred for 15 minutes while being allowed to warm toroom temperature, whereupon a solution of Boc-HphH (0.707 g, 2.68 mmol)in THF (5 mL) was added. The mixture was stirred for 20 minutes. 1M HCl(15 mL) was added. The product was extracted into ethyl acetate (40 mL),washed with saturated aqueous NaHCO₃ (15 mL), brine (15 mL), dried overMgSO₄, filtered, and evaporated to dryness. The product,(S)-(E)-(N-phenylalanine silylethylester)-4-tert-butoxycarbonylamino-6-phenyl-2-hexenamide(Boc-HphVAM-PheOSET) was crystallized from CH₂Cl₂ and 1:1 ether/hexane.Yield=0.78 g (53%). TLC analysis (30% ethyl acetate/hexane) showed aretention factor of 0.35.

To a solution of Boc-HphVAM-PheOSET (1.80 g, 3.25 mmol) in CH₂Cl₂ (1 mL)were added 8 mL of a 4.0 M solution of HCl in dioxane. The mixture wasstirred for 60 minutes, whereupon the solvents were removed underreduced pressure. The residue was pumped to a pale yellow foamy solid,(S)-(E)-(N-phenylalanine silylethyl ester)-4-amino-6-phenyl-2-hexenamide(HCl.HphVAM-PheOSET). Yield=1.40 g (88%).

To a solution of Mu-PheOH (0.797 g, 2.86 mmol) in THF (10 mL) at −10° C.was added 4-methylmorpholine (0.315 mL, 2.86 mmol), followed by isobutylchloroformate (0.371 mL, 2.86 mmol). The mixed anhydride was stirred for5 minutes, whereupon a solution of HCl.HphVAM-PheOSET (1.40 g, 2.86mmol) in THF (5 mL) was added.

4-methylmorpholine (0.315 mL, 2.86 mmol) was added. The mixture wasstirred for 75 minutes. Ethyl acetate (40 mL) was added. The mixture waswashed with 1M HCl (15 mL), saturated aqueous NaHCO₃ (15 mL), brine (10mL), dried over MgSO₄, filtered, and evaporated to dryness, giving 1.9 g(93%) of the product, (S)-(E)-(N-phenylalanine silylethylester)-4-(4-morpholinecarbonylphenylalanyl)amino-6-phenyl-2-hexenamide(Mu-Phe-HphVAM-PheOSET). TLC analysis (50% ethyl acetate/CH2Cl2)revealed a retention factor of 0.32.

To a solution of Mu-Phe-HphVAM-PheOSET (1.59 g, 2.23 mmol) in THF (10mL) were added ˜2 g of 3 Å molecular sieves, followed by 2.23 mL of a1.0 M THF solution of tetrabutylammonium fluoride. The mixture wasstirred at room temperature overnight. Ethyl acetate (50 mL) was added.The solution was filtered through Celite, washed with 1M HCl (20 mL),brine (20 mL) dried over MgSO₄, filtered, and evaporated to dryness. Theresidue was dissolved in CH₂Cl₂ (10 mL) and the solution was poured intoether (300 mL). The precipitate was collected on a Buchner funnel,washed with ether (2×20 mL), and pumped dry to give 0.84 g (61%) ofMu-Phe-HphVAM-PheOH as a white solid.

The melting point was determined to be 95-98° C.

TLC analysis (10% CH₃OH/CH₂Cl₂) showed a retention factor of 0.05-0.18(yellow stain with bromocresol green indicating acidic functionality.)

Mass spectroscopy (FAB, low resolution): calculated for C₃₅H₄₀N₄O₆,(m+H)=613, found weak probable molecular ion cluster centered atm/z=614.

Example 14 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone

Synthesis of(S)-(E)-3-tert-butoxycarbonylamino-4-tertbutoxycarbonyl-1-methylsulfonyl-1-butene,abbreviated herein as (Boc-Asp(Ot-Bu)VSMe, was as follows. The aldehpydeof Boc-Asp(Ot-Bu) was prepared in a slightly modified version of themanner of Fehrentz, supra, by the conversion to the N₂0-dimethyl amidein quantitative yield. Reduction with lithium aluminum hydride (0.5 moleequivalents) reduced the amide in the presence of the tertbutyl ester in88% yield. Sodium hydride (0.633 g of a 60% mineral oil suspension,16.58 mmol) was added to a solution of diethyl methylsulfonylmethylenephosphonate (3.5 g, 15.2 mmol) in THF (50 ml) at 0° C. The mixture waswarmed to 25° C. over 60 minutes. Boc-AspH(β-O-t-Bu) (3.78 g, 13.82mmol) was added as a solution in THF (15 mls). The mixture was stirredfor 60 minutes. 50 mls of 1 M HCl was added. The product was extractedwith 100 mls of ethyl acetate, washed with 50 mls saturated aqueousNaHCO₃, 50 mls of brine, dired over CaCl₂, filtered, and evaporated todryness. The residue was purified by column chromatography (30-60% ethylacetate in hexane, gradient elution) to afford the product, (1.88 gm,39%), that could be crystallized from ether and hexane.

TLC (30% ethyl acetate/hexane): R_(f)=0.25. ¹H NMR (CDCL₃): 1.43 (18H,2×s*, t-Bu CH₃'s); 2.55 (2H, 2×dd, J=16,7 Hz, CH₂COOt-Bu); 2.91 (3H, s,CH₃); 4.69 (1H, m, CHNH); 5.38 (1H, m, NHCH); 6.50 (1H, dd, J=15,2 Hz,trans SO₂CH═CH); 6.86 (1H, J=5, 15 Hz, trans CH═CHSO₂).

Example 15 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone

(S)-(E)-3-amino-4-tert-butoxycarbonyl-1-methylsulfonyl-1-butene,abbreviated TsOH.Asp(Ot-Bu)VSMe was synthesized as follows. A solutionof anhydrous p-toluenesulfonic acid (0.95 g, 5.58 mmol) (commerciallyavailable as the hydrate from Aldrich) in 1 ml ether was added to asolution of Boc-Asp(Ot-Bu)VSME (0.41 g, 1.17 mmol) in 1:1dichloromethane/ether (4 ml). The mixture was stirred at roomtemperature overnight. 15 ml of ether was added. The product, aprecipitate, was filtered, washed with 20 mls ether, and dried in vacuoto give 0.49 g (99%) of the product. By using p-toluenesulfonic acid,the Boc group was selectively removed in the presence of the less labiletert-butyl ester.

Example 16 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone

Synthesis of(S)-(E)-3-tert-butoxycarbonylamino-4-tert-butoxycarbonyl-1-phenylsulfonyl-1-butene,abbreviated Boc-Asp(Ot-Bu)-VSPh was as follows. Sodium hydride (0.489 gof a 60% mineral oil dispersion, 12.23 mmol) was added to a solution ofPMSP (3.58 g, 12.23 mmol) in 50 mL of THF at 0° C. The mixture wasstirred for 15 minutes. A solution of Boc-AspH(β-Ot-Bu), prepared asdescribed above (3.04 gm, 11.12 mmol) in 10 mls THF was added. Themixture was stirred for 1 hour, whereupon 30 mls of 1 M HCl was added.The product was extracted with ethyl acetate (100 ml), and washed with50 mls of saturated aqueous NaHCO₃, 30 mls brine, dired over MgSO₄.filtered, and evaporated to dryness. Chromatography on silica gel(20-30% ethyl acetate/hexane, gradient elution) afforded 2.07 g 45%) ofthe product.

TLC=R_(f)=0.31.

Example 17 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone

Synthesis of (S)-(E)-3-amino4-tert-butoxycarbonyl-1-phenylsulfonyl-1-butene-p-toluenesulfonate,abbreviated TsOH.Asp(Ot-Bu)VSPh, was as follows. A solution of anhydrousp-toluenesulfonic acid (1.0 g, 5.87 mmol) in 2 mls ether was added to asolution of Boc-Asp(Ot-Bu)VSPh (0.72 g, 1.75 mmol) in 2 mls ether. Themixture was stirred at room temperature overnight, then diluted withether (25 mls). The white precipitate, was filtered, washed with ether,and dried in vacuo to give 0.80 g (95%) of the product.

Example 18 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone

Synthesis of(S)-(E)-3-amino-4-hycroxylcarbonyl-1-phenylsulfonyl-1-butene-p-toluenesulfonate,abbreviated HCl.AspVSPh was as follows. To a solution ofBoc-Asp(Ot-Bu)VSPh (0.32 g, 0.778 mmol) in 2 mls ether was added 2 misof 4.0 M HCl in dioxane (Aldrich). The mixture was stirred at roomtemperature overnight. 50 mls ether was added. The supernatant wasdecanted, and the residue was precipitated from methanol/ether,filtered, and dried in vacuo to give 0.20 g (88%) of product. By usingHCl, both Boc and t-butyl ester groups were thus removed in onereaction.

Example 19 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone

Synthesis of(E)-3-acetyltyrosylvalylalanylamino-4-tert-butoxycarbonyl-1-phenylsulfonyl-1-butene,abbreviated Ac-Tyr-Val-Ala-Asp(Ot-Bu)VSPh, was as follows.Ac-Tyr-Val-AlaOH was prepared by standard peptide chemistry and coupledvia mixed anhydride chemistry to TsOH.Asp(Ot-Bu)VSPh. The proton NMRspectrum (in CD₈OD) indicated a singlet at 1.18 ppm, integrating to 9H(t-butyl ester), a singlet at 1.75 integrating to 3H (CH₈ of acetate atN-terminus). Signals for the alkene group hydrogens are obscured by thearomatic Tyr protons at 6.6-7.2 ppm. The sulfonate aromatic protons (5H)absorbed at 7.6-7.9 ppm. The presence of the remaining residues in theNMR spectrum was clarified, in part, by removal of the t-butyl ester andspectroscopic analysis (infra).

Example 20 Synthesis of a Cysteine Protease Inhibitor with VinylogousSulfones

Synthesis of(E)-3-acetyltyrosylvalylalanylamino-4-hydroxycarbonyl-1-phenylsulfonyl-1-butene,abbreviated Ac-Tyr-Val-Ala-AspVSPh was as follows. Trifluoroacetic acid(0.5 mL) was added to Ac-Tyr-Val-Ala-Asp(O-t-Bu) VSPh (45 mg, 66.7μmol). The mixture was permitted to stand at room temperature overnight.Ether (20 mL) was added. The precipitate was chilled to −20° C.,filtered, washed with ether (10 mL), and dried in vacuo.

¹H NMR (CD₃OD/DMSO-d⁶) indicated Ala and Val residues (0.8-1.2 ppm). Thet-butyl group singlet at 1.18 ppm was no longer present. The presence ofthe vinyl sulfone group was indicated as a doublet (J=15 Hz) at 6.66ppm, partially obscured by the doublet of the Tyr-aromatic residue, andas a doublet of doublets (J=15,5 Hz) at 6.91 ppm. The other Tyr-aromaticresidue was seen as a doublet at 7.01 ppm. The sulfone aromatic protons(5H) were seen between 7.6 and 7.85 ppm.

The above examples demonstrate the feasibility of chemical manipulationof aspartic acid derivatives with the Asp side chain protected anddeprotected. By using toluenesulfonic acid and trifluoroacetic acid atthe appropriate stages of the synthesis, α,β-unsaturated sulfones[EWG'S] can be prepared as ICE inhibitors bearing Asp as the P₁ activesite side chain moiety.

Example 21 Synthesis of a Cysteine Protease Inhibitor with a VinylogousSulfone

Synthesis of (S)-(E)-3-(4-morpholine-carbonylleucyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene, abbreviated Mu-Leu-HphVSPh,was as follows. Preparation of Mu-Leu-Hph was achieved by coupling ofHCl.HphVSPh (described in the preparation of Mu-Phe-HphVSPh) withleucine morpholine urea (Mu-LeuOH). Mu-LeuOH was prepared in two steps,namely by treating the leucine benzyl ester p-toluenesulfonate (Bachem)with 4-morpholine chloride (Aldrich) in the presence of triethylamine(Aldrich) to give Mu-LeuOBzl in 99% yield. Hydrogenolysis over 5%palladium/charcoal in ethyl acetate afforded the desired product inquantitative yield.

Example 22 Inhibition of Cathepsin B

Stock solutions of the various inhibitors (10 mM) were made in DMF;dilutions were made in the same solvent. Cathepsin B from humanplacenta, obtained from Sigma, was approximately 10 nM in the assay. Theassay buffer contained 50 mM phosphate at pH 6.2, 2.5 mM dithiothreitol(DTT), and 2.5 mM ethylenediaminetetraacetic acid (EDTA). The substratefor cathepsin B was 50 μM Z-Arg-Arg-AMC(carbobenzyloxyarginylarginylaminomethylcoumarin). The assays wereinitiated at 25° C. by addition of enzyme to 2 mL of assay buffercontaining the substrate and the various inhibitors (30-0.001 (μM). Theincrease in fluorescence at 460 nm with excitation at 380 nm wasfollowed with time, and the assay was linear throughout the rangeobserved. Inactivation of cathepsin B was indicated by a downward curvein the increase in fluorescence with time. In the absence of inhibitors,the enzymatic production of free AMC was linear throughout the timecourse of inactivation examined. Data points were collected over atleast 3 half-times of inactivation, and in duplicate. The inhibitionprogress curves were analyzed using non-linear curve fitting software tocompute the k_(obs) values. Approximate second-order inactivationconstants (k_(obs)/[I]) were calculated for all compounds. For selectedinhibitors, plots of 1/[I] vs. 1/k_(obs) yielded the true constantsk_(irr), K_(I), and k_(irr)/K_(I), and were corrected for the presenceof substrate by dividing K_(I)(app.) by {1+[S]/K_(m)}.

The results are shown in Table 2.

TABLE 2 k/K_(I) or k_(obs)/[I]* Inhibitor (M⁻¹ sec⁻¹) k_(irr), sec⁻¹K_(I), μM Mu-Tic-HphVSPh 0 — — Ac-Leu-Leu-NleVSPh 0 — —Ac-Leu-Leu-MetVSPh 0 — — Mu-DL-Fam-HphVSPh 0 — binds Boc-D(O-tBu)VSMe 0— — Ac-Tyr-Tyr-Val-Ala- 0 — — AspVSMe Mu-Phe-HphVAMBzl <100 — —Mu-Arg-ARgVSPh.2HBr 26- 0.040 150 Mu-Phe-AlaVSMe 300 0.19 ˜1000Boc-Np-HphVSPh 400* — — Mu-Phe-HphVA 450 0.038 85 Mu-Phe-HphVAM.PheOH600* — — Mu-Phe-LysVEOEt.HBr 650* — — Z-Leu-Leu-TyrVSPh 730* — —MeOSuc-Phe-HphVSPh 870* — — Boc-Tyr(I₂)-HphVSPh 1400* — —Mu-Phe-Met(O₂)VSPh 1,600* — — Mu-Phe-Lys(Z)fVSPh 2,000* — —Mu-Phe-OBSer-VSPh 2900 0.14 47 Mu-Phe-Hph-VSPh 3100 0.061 19Mu-Leu-Hph-VSPh 4250 — — Mu-Phe-Lys(Z)VEOEt 4100 0.13 32 Mu-fPhe-HphVSPh4800* — — Mu-Phe-HpHVSMe 5100 0.13 26 Mu-Phe-MetVSPh 7,000Mu-Phe-Nle-VSPh 7,600 Mu-Phe-ArgVEOEt.HBr 8200 0.16 19 Mu-Phe-HphNS9,200 0.042 4.6 Mu-Phe-HphVEOEt 10,100 0.19 19 Mu-Phe-LysVSPh.HBr 11,700— — (impure) Mu-Phe-LysVSPh.HBr 11,300* — — (pure) Mu-Phe-ArgVSPh.HBr12,300 0.15 12 Mu-Phe-HphVSPhBr 15,500* — — Mu-Phe-HphVSPh 16,400 0.1711 Mu-Tyr-HphVSPh 20,000* — — Mu-Phe-HphNS 21,300 0.60 28Mu-Phe-Hph-VSPh 22,600 0.12 5.3 Mu-Phe-Lys(Z)VSPh 39,200 0.18 4.7Mu-Phe-HphVSNp 63,700 0.087 1.4 Mu-Np-HphVSPh 86,000* — — Mu-Np-HphVSNp184,000* — — Mu-Tyr(I₂)-HphVSPh 39,200 0.18 4.7

Example 23 Inhibition of Cathepsin L

Stock solutions of the various inhibitors (10 mM) were made in DMF;dilutions were made in the same solvent. Cathepsin L from rat liver,(see Mason et al., Biochem. J. 226:233-241 (1985)) was approximately 1nM in the assay. The assay buffer contained 50 mM phosphate at pH 6:2,2.5 mM dithiothreitol (DTT), and 2.5 mM ethylenediaminetetraacetic acid(EDTA). The substrate for cathepsin L was 5 μM Z-Phe-Arg-AMC(carbobenzyloxyphenylalanylarginylaminomethylcoumarin). The assays wereinitiated at 25° C. by addition of enzyme to 2 mL of assay buffercontaining the substrate and the various inhibitors (30-0.001 μM). Theincrease in fluorescence at 460 nm with excitation at 380 nm wasfollowed with time. Inactivation of cathepsin L was indicated by adownward curve in the increase in fluorescence with time. In the absenceof inhibitors, the enzymatic production of free AMC was linearthroughout the time course of inactivation examined. Data points werecollected over at least 3 half-times of inactivation. The inhibitionprogress curves were analyzed using non-linear curve fitting software tocompute the k_(obs) values. Approximate second-order inactivationconstants (k_(obs)/[I]) were calculated for all compounds. For selectedinhibitors, plots of 1/[I] vs. 1/k_(obs) yielded the true constantsk_(irr), K_(I), and k_(irr/K) _(I) and were corrected for the presenceof substrate by dividing K_(I)(app.) by (1+[S]/Km).

The results are shown in Table 3.

TABLE 3 k/K_(I) or k_(obs)/[I]* Inhibitor (M⁻¹ sec⁻¹) k_(irr), sec⁻¹K_(I), μM Boc-Tic-HphVSPh 0 — — Mu-DL-Fam-HphVSPh 0 — bindsBoc-Asp(O-tBu)VSMe 0 — — Ac-Tyr-Val-Ala-AspVSPh 0 — — Mu-Phe-PheVSMe100* — — Mu-Phe-HphVAmBzl 2800* — — Mu-Phe-LysVEOEt.HBr 3500 0.085 24.2Mu-Phe-HphVPEt 4100 0.16 39 Mu-Phe-HphVPhe 8,600 0.032 3.7Mu-Phe-HpHVSMe 8700* — — Mu-Tic-HphVSPh 12,600* — — Mu-Phe-Lys(Z)VEOEt13,900 0.87 6.2 Mu-Phe-D-HphVSPh 4,500* — — Mu-Leu-HphVSPh 28,700* — —Mu-Phe-Ser(OBzl)VSPh 42,700 0.20 4.6 MeOSuc-Phe-HphVSPh 47,000* — —Mu-Phe-HphVEOEt 47,500 0.10 2.2 Mu-Phe-ArgVEOEt.HBr 56,900 0.18 3.1Mu-Phe-HphNS 57,400 — — Mu-Phe-ValVSPh 74,000 0.19 2.6Boc-Tyr(I₂)-HphVSPh 105,000* — — Mu-Phe-LysVSPh.HBr 110,000 0.16 1.4Mu-Phe-Lys(Z)VSPh.HBr 120,000 0.45 3.8 Mu-Phe-NleVSPh 188,000 — —Mu-Tyr(I₂)HphVSPh 190,000* — — Mu-Phe-LysVSPh.HBr 220,000* — —Mu-Phe-HphVSPh 224,000 0.24 1.0 Mu-Phe-HphVSPh (pure) 260,000* — —Mu-Phe-Lys(Bz)VSPh 210,000* — — Mu-Phe-Met(O₂)VSPh 250,000 — —Mu-Phe-MetVSPh 250,000 — — Mu-Phe-Hph-VSPh 201,000 0.23 1.1Mu-Phe-ArgVSPh.HBr 349,000 0.67 1.9 Mu-Leu-HphVSPh 490,000 0.40 0.81Mu-Tyr-HphVSPh 700,000* — — Ac-Leu-Leu-MetVSPh 880,000 — —Ac-Leu-Leu-NleVSPh 930,000 — — Mu-Np-HphVSPh 1,000,000* — —Ac-Leu-Leu-Met(O₂)VSPh 1,500,000 — — Boc-Np-HphVSPh 1,600,000* — —Mu-Np-HphVSNp 9,200,000 0.14 0.018

Example 24 Inhibition of Cathepsin S

Stock solutions of the various inhibitors (10 mM) were made in DMF;dilutions were made in the same solvent. Cloned cathepsin S, (see Brömmeet al., J. Biol. Chem. 268(&): 4832-4838 (1993)) was less than 1 nM inthe assay. The assay buffer contained 100 mM phosphate at pH 6.5, 0.01%Triton, 2.5 mM dithiothreitol (DTT), and 2.5 mMethylenediaminetetraacetic acid (EDTA). The substrate for cathepsin Swas 10 μM Z-Val-Val-Arg-AMC(carbobenzyloxyvalinylvalinylarginylaminomethylcoumarin). The assayswere initiated at 25° C. by addition of enzyme to 2 mL of assay buffercontaining the substrate and the various inhibitors (30-0.001 μM). Theincrease in fluorescence at 460 nm with excitation at 380 nm wasfollowed with time. Inactivation of cathepsin S was indicated by adownward curve in the increase in fluorescence with time. In the absenceof inhibitors, the enzymatic production of free AMC was linearthroughout the time course of inactivation examined. Data points werecollected over at least 3 half-times of inactivation. The inhibitionprogress curves were analyzed using non-linear curve fitting software tocompute the k_(obs) values. Approximate second-order inactivationconstants (k_(obs)/[I]) were calculated for all compounds. For selectedinhibitors, plots of 1/[I] vs. 1/k_(obs) yielded the true constantsk_(irr), K_(I), and k_(irr)/K_(I), and were corrected for the presenceof substrate by dividing K_(I)(app.) by {1+[S]/Km}.

The results are shown in Table 4.

TABLE 4 k/K_(I) or k_(obs)/[I]* Inhibitor (M⁻¹ sec⁻¹) k_(irr), sec⁻¹K_(I), μM Boc-Tic-HphVSPh 0 — — Mu-Phe-HphVAM-PheOH 10,600* — —Mu-Phe-Lys(Z)fVSPh 10,700* — — Mu-Phe-HphVAMBzl 10,800* — —Mu-Phe-HpHVPEt 11,200 0.40 36 Mu-Leu-Leu-TyrVSPh 25,100* — —Mu-Phe-Ala-VSMe 26,000* — — HCl.Phe-HphVSPh 35,000* — —Mu-DL-Fam-HphVSPh 39,000 0.54 14 Mu-Phe-HphNS 40,300 0.033 0.81Mu-Phe-D-HphVSPh 68,500* — — Mu-Phe-Lys(Z)VEOEt 100,000 0.13 1.3Ac-Leu-Leu-TyrVSPh 100,000* — — Mu-Phe-HphNS 140,000 0.08 0.58Boc-Tyr(I₂)HphVSPh 210,000* — — Z-Leu-Leu-TyrVSPh 280,000* — —Mu-Phe-ValVSPh 290,000 0.039 0.13 Mu-Tic-HphVSPh 630,000* — —MeOSuc-Phe-HphVSPh 740,000* — — DimSam-Phe-HphVSPh 920,000* — —Mu-Phe-HpHVSMe 1,200,00 0.10 0.088 Mu-Phe-Ser(OBzl)VSPh 1,240,000 0.0920.074 Mu-Phe-ArgVSPh.HBr 2,000,000 0.031 0.016 Mu-Phe-Nle-VSPh2,300,000* — — Mu-Phe-Lys(Bz)VSPh 2,500,000* — — Mu-Phe-Lys(Z)VSPh2,600,000 0.14 0.054 Mu-Phe-Met(O₂)VSPh 2,800,000 — — Z-Leu-PheVSPh3,000,000* — — Mu-Phe-MetVSPh 4,000,000 — — Boc-Np-HphVSPh 4,100,000* —— Mu-Tyr-HphVSPh 4,200,000* — — Mu-Phe-HphVSNp >5,400,000* — —Mu-Phe-HphVSPh 7,700,000 0.085 0.011 Mu-Tyr(I₂)HphVSPh >7,300,000* — —Mu-Phe-LysVSPh 10,700,000* — — Mu-fPhe-HphVSPh 13,300,000* — —Mu-Phe-Hph-VSPh 6,500,000 0.15 0.023 Mu-Np-HphVSNp 56,000,000 0.10 .0018Mu-Leu-HphVSPh 26,300,000 0.16 .00590

Example 25 Inhibition of Cruzain

Inhibition of cruzain, from T. cruzi, (see Eakin et at., J. Biol. Chem.268(9): 6115-6118 (1993)) proceeded exactly as for cathepsin L, outlinedabove, using an enzyme concentration of 1 nM.

The results are shown in Table 5.

TABLE 5 k/K_(I) or k_(obs)/[I]* Inhibitor (M⁻¹ sec⁻¹) k_(irr), sec⁻¹K_(I), μM Ac-Tyr-Val-Ala- 0 — — AspVSPh Mu-Tic-HphVSPh 0 — —Mu-Phe-AlaVSMe 700* — — Boc-ASp(O-tBu)VSMe 800* — — Mu-ARg-Arg-VSPh2,100* — — Mu-Phe-Ser-(OBzl)VSPh 12,800 — — HCl.Phe-HphVSPh 14,600* — —Mu-DL-Fam-HphVSPh 18,100* — — Mu-Phe-HphVSMe 22,000* — — Mu-Phe-ValVSPh28,000* — — Boc-Tic-HphVSPh 30,000 — — MeOSuc-Phe-HphVSPh 34,000* — —Mu-Phe-Lys(Z)VSPh 44,500 — — Z-Leu-Leu-TyrVSPh 45,000* — — Mu-Phe-HphVNS58,000* — — Mu-Phe-Lys(Bz)VSPh 60,000* — — Mu-Phe-HphVAMBzl 70,200* — —Mu-Phe-NleVSPh 77,000 — — Mu-Phe-HphVEOEt 80,400* — — Mu-Phe-ArgVSPh91,400 — — Acc-Leu-Leu- 104,000 — — Met(O₂)VSPh Ac-Leu-Leu-MetVSPh110,000 — — Mu-Phe-MetVSPh 111,000 — — Mu-Tyr(I₂)HphVSPh 114,000* — —Ac-Leu-LeuNleVSPh 133,000 — — Mu-Phe-HphVSPh 134,000 0.43 3.2Mu-Phe-LysVSPh 149,000* — — Mu-Phe-Met(O₂)VSPh 160,000 — —Boc-Np-HphVSPh 210,000* — — Mu-Np-HphVSPh 218,000 — — Boc-Tyr(I₂)HphVSPh280,000* — — Mu-Tyr-HphVSPh 297,000* — — Mu-Phe-HphVSNp 740,000 0.08-0.11 Mu-Np-HphVSNA 1,770,000 0.044 0.025 Mu-Leu-HphVSPh 213,000 0.110.52

Example 26 Stability of Selected Inhibitors Toward Glutathione

The inhibitors (5 mM) were incubated with glutathione (GSH) (0.3 mM) inphosphate buffer (50 mM) at pH=6.2 containing 15% DMF at 20° C.Periodically, 50 μL samples were added to 1 mL of buffer and treatedwith 10 μL of 30 mM Ellmans reagent and the absorbance measured at 412nm.

After 24 hours there was no measurable reaction between Mu-Phe-HphVSMeor Mu-Phe-HphVPEt with GSH. The second order rate constant for the lossof GSH was measured to be 5.5×10⁻⁴ M⁻¹s⁻¹ for Mu-Phe-LysVSPh. The vinylester analog Mu-Phe-ArgVEOEt was 10 times more reactive at 5.3×10⁻³M⁻¹s⁻¹. However, these compounds react at a minimum of 10⁶-10⁹ timesfaster at the active sites of cysteine proteases than with GSH.

Example 27 Selectivity for Cysteine Proteases Versus Serine Proteases

Boc-Ala-PheVSPh (100 μM, in 100 mM TRIS buffer at pH=7.5) did not bindto chymotrypsin, nor did it inactivate chymotrypsin after 1 hour. Thesame was true for MeOSuc-Ala-Ala-Pro-ValVSPh (100 μM, in 100 mM TRISbuffer at pH=7.5) with elastase.

Example 28 Synthesis of a Cysteine Protease Inhibitor with a 6 MemberedHomocyclic Substituted Aromatic Ring as the EWG

Synthesis of Boc-phenylalanyl-homophenylalanyl-p-nitrostyryl,abbreviated herein as Boc-Phe-Hph-VNS, was as follows. Triphenylphoshine(10 gm, 38.1 mmmol) and 4-chloro-p-nitrophenyl (6.54 g, 38.1 mmol) weredissolved in 200 ml of THF and heated at reflux for 2 days. The whitesolid was filtered and washed with 200 ml diethyl ether and placed invacuo for 16 hours.

p-nitromethylphenyltriphenylphosponium chloride (1.20 g, 2.77 mmol) wasdissolved in 50 ml H₂O and 1.4 ml of 2 M, (2.77 mmol) sodium hydroxidewas added. An immediate glassy red colored solid was formed, and the H₂Olayer was extracted with 100 ml of toluene. The organic layer was driedover K₂CO₃, filtered and concentrated to dryness. The resulting redsolid was dissolved in 100 ml of THF and 0.73 g (2.77 mmol) ofBoc-homophenylalanyl aldehyde was added. The solution turned yellow overtime. The reaction was complete after 16 hours. The reaction mixture wasdiluted with 250 ml of CH₂Cl₂ and washed with 200 ml of 1 M HCl, and 200ml of saturated aqueous NaHCO₃, dried over MgSO₄, filtered andconcentrated to dryness. The residue was dissolved in 20 ml of a 20%ETOAc/hexane solution and eluted down a silica gel column with 20%ETOAc/hexane solution. 25 ml fractions were collected. Fractions 6-12were combined and concentrated to dryness. 340 mg of a 50/50 mixture ofcis-trans configuration of Boc-homophenylalanyl-p-nitrostyryl wasrecovered.

The 340 mg (0.89 mmol) of Boc-homophenylalanyl-p-nitrostyryl wasdissolved in 10 ml of HCl in dioxane (4 M) and left to stir under adrying tube for 30 minutes and then concentrated to a yellow foam.

0.29 g (0.98 mmol) Boc-phenylalanine was dissolved in 50 ml of THF and216 μl (1.96 mmol) of N-methylmorpholine was added. The reaction wasthen stirred at 0° C. for 5 minutes under argon and 129 μl (0.98 mmol)of isobutylcholorformate was added, and a white precipitate began toform. After 5 minutes, the HCl salt of homophenylalanyl-p-nitrostyryl(284 mg, 0.98 mmol) in 10 ml of DMF was added and the reaction wasallowed to warm up to room temperature over 16 hours. The reaction wasthen concentrated and then diluted with 200 ml of CH₂Cl₂ and washed with200 ml of 1 M HCl and 200 ml of saturated aqueous NaHCO₃, dried overK₂CO₃, filtered and concentrated to dryness. The product was dissolvedin 10 ml EtOAc and diluted with 20 ml of diethyl ether, and a whitesolid was filtered and dried in vacuo. 200 mgs of the resulting product,Boc-phenylalanyl-homophenylalanyl-p-nitrostyryl, was recovered as a50/50 mixture of cis and trans configuration as determined by ¹H NMR,and by TLC: 30% EtOAc/hexane, with bromo creoso green stain: R_(f) ofthe two forms, cis and trans, was 0.48 and 0.54.

Example 29 Assay of a Cysteine Protease Inhibitor with a 6 MemberedSubstituted Homocyclic Ring as the EWG

The cysteine protease inhibitor made in Example 15,Boc-phyenylalanylhomophenylalanylvinyl-p-nitrostyryl, was tested againstcathepsin S, as outlined in Example 12. The inhibitor showed ak_(obs)/[I] value of 40,100 M⁻¹sec⁻¹.

Example 30 Synthesis of a Cysteine Protease Inhibitor with aDienylsulfone as the EWG

Synthesis of(S)-(E,E)-5-(4-morpholinecarbonylphenylalanyl)amino-7-phenyl-1-phenylsuflonyl-1,3-heptadiene(Mu-Phe-Hph-DIESPh). a) Diethyl phosphonoacetyl N,O-dimethylhyroxamide{(EtO)₂POCH₂CON(Me)OMe} was prepared in 82% yield from diethylphosphonoacetic acid and N,O-dimethylhydroxylamine hydrochloride in thepresence of dicyclohexylcarbodiimide and triethylamine. b) To a solutionof {(EtO)₂POCH₂CON(Me)OMe (1.13 g, 4.71 mmol) in THF (25 mL) at 0° C.was added sodium hydride (0.188 g, 4.71 mmol as a 60% mineral oildispersion. After 15 minutes, a solution of Boc-homophenylalaninal (1.24g, 4.71 mmol) in THF (5 mL) was added. The mixture was stirred for 30minutes. 1M HCl (50 mL) was added. The intermediate,(S)-(E)-4-tert-butoxycarbonylamino-6-phenyl-2-hexenoylN,O-dimethylhydroxamide (Boc-HphVAMN(Me)OMe) was extracted with ethylacetate (50 mL), washed with saturated aqueous sodium bicarbonate (50mL) and brine (50 mL), dried over MgSO₄, filtered, and the solvent wasremoved under reduced pressure. c) The resulting oil was dissolved inTHF (25 mL) and cooled to 0° C. Lithium aluminum hydride (5 mL of a 1.0MTHF solution) was added. The solution was stirred for 20 minutes. Water(5 mL) was carefully added, followed by 1M HCl (30 mL). The nextintermediate, (S)-(E)-4-tert-butoxycarbonylamino-6-phenyl-2-hexenal(Boc-HphVAl) was extracted into ethyl acetate (50 mL), washed withsaturated aqueous sodium bicarbonate (30 mL) and brine (30 mL), driedover MgSO₄, filtered, and the solvent was removed under reducedpressure. TLC: (30% ethyl acetate/hexane) R_(f)=0.31. The crudematerial, weighing 1.36 g, was used immediately in the next step. d) Toa solution of PSMP (1.38 g, 4.71 mmol) in THF (20 mL) at 0° C. was addedsodium hydride (0.188 g of a 60% mineral oil dispersion, 4.71 mmol). Themixture was stirred for 15 minutes, whereupon a 5 mL THF solution ofBoc-HphVAl from the previous step was added. The mixture was stirred for40 minutes. 1M HCl (25 mL) was added. The intermediate,(S)-(E,E)-5-tert-butoxycarbonylamino-7-phenyl-1-phenylsulfonyl-1,3-heptadiene(Boc-Hph-DIESPh), was extracted with ethyl acetate (25 mL), washed withsaturated aqueous sodium bicarbonate (25 mL), brine (25 mL), dried overMgSO₄, filtered, and the solvent was removed under reduced pressure. Theresidue was crystallized from CH₂Cl₂/ether/hexane to give 0.80 g (39%)from Boc-homophenylalaninal. TLC: (30% ethyl acetate/hexane) R_(f)=0.16.e) To a solution of Boc-HphDIESPh (0.80 g, 1.87 mmol) in ether/CH₂Cl₂ (3mL, 2:1) was added a solution of anhydrous p-toluenesulfonic acid (0.80g, 4.70 mmol) in ether (3 mL). The mixture was stirred at roomtemperature overnight. An additional 5 mL aliquot of CH₂Cl₂ was addedand stirring permitted to continue for another 24 hours. Ether (80 mL)was then added. The solids were filtered, washed with ether (2×20 mL),and dried in vacuo to give 0.69 g (74%) of the intermediate,(S)-(E,E)-5-amino-7-phenyl-1-phenylsulfonyl-1,3-heptadiene4-toluenesulfonate (TsOH.HphDIESPh). f) To a solution of Mu-PheOH (0.334g, 1.20 mmol) in THF (7 mL) at −10° C. were added 4-methylmorpholine(0.132 mL, 1.20 mmol) and isobutyl chloroformate (0.156 mL, 1.20 mmol).The mixed anhydride was stirred for 5 minutes, whereupon TsOH.HphDIESPh(0.60 g, 1.20 mmol) was added, followed by 4-methylmorpholine (0.132 mL,1.20 mmol). The reaction mixture was stirred for 45 minutes. Ethylacetate (40 mL) was added. The solution was washed with 1M HCl,saturated aqueous sodium bicarbonate, and brine (5 mL each), dried overMgSO₄, filtered, and the solvent was removed under reduced pressure. Theproduct, Mu-Phe-HphDIESPh, formed an oil on attempts to crystallize it.The supernantant was discarded and the residue was dried in vacuo,resulting in a foam (0.43 g, 61%).

TLC (50% ethyl acetate/CH₂Cl₂) R_(f)=0.29. ¹H NMR (CDCl₃): 1.6-1.95 (2H,m, CH₂CH₂C₆H₅); 2.57 (2H, m, CH₂CH₂C₆H₅); 3.03 (2H, 2×dd, PhCH₂CH); 3.25(4H, NCH₂CH₂O); 3.59 (4H, m, NCH₂CH₂O); 4.43 (1H, m*, CHNH (Hph)); 4.48(1H, q*, J=6 Hz, CHNH (Phe)); 5.05 (1H, d, J=6 Hz, NHCH (Mu)); 5.86 (2H,m*, CH═CH); 6.09 (1H, d, J=6 Hz, NHCH); 6.26 (1H, d, J=12 Hz, SO₂CH═CH);7.04-7.92 (16H, m, 15×aromatic and one CH═CH). The presence of a smallerdoublet at 4.96 ppm, assigned as an NHCH peak corresponding to themorpholine urea, suggests that the all-trans configuration anticipatedby performance of successive Wadsworth-Emmons reactions may in thissequence result in at least one of the double bonds in a minor componentof the product being of cis configuration.

Example 31 Synthesis of a Cysteine Protease Inhibitor with a VinylSulfone as the EWG with a Fluoro Moiety as an Additional EWG Attached tothe Same Carbon of the Alkene Bond

Synthesis of (S)-(E,Z)-1-fluoro-3-(4-morpholinecarbonyl-phenylalanyl)amino-5-phenyl-1-phenylsulfonyl-1-pentene (Mu-Phe-Hph-VSPh-VF). a) To asolution of diethyl phenylsulfonylmethylenephosphonate (5.06 g, 17.31mmol) in THF (100 mL) at 0° C. was added sodium hydride (0.83 g of a 60%mineral oil dispersion, 20.77 mmol). The mixture was stirred for 20minutes. N-fluorodiphenylsulfonimide (8.73 g, 27.69 mmol) was added as asolid. The mixture was stirred at room temperature overnight. Thefluorinated Wadsworth-Emmons reagent,diethylphosphonylphenylsulfonylfluoromethane (PSMP-F) was isolated bypartitioning between ethyl acetate (100 mL) and 1M HCl (100 mL). Theorganic phase was washed with saturated aqueous sodium bicarbonate (100mL), brine (50 mL), dried over MgSO₄, filtered, and the solvent wasremoved under reduced pressure. The residue was purified by columnchromatography (60-200 mesh silica gel, 0-20% ethyl acetate/CH₂Cl₂,gradient elution) to give 2.03 g (38%) of PSMP-F. TLC: (20% ethylacetate/CH₂Cl₂) R_(f)=0.5. ¹H NMR (CDCl₃): 1.37 (6H, 2×t, 2×CH₃); 4.26(4H, m, 2×CH₂OP); 5.39 (1H, dd, CHFP); 7.57-8.03 (5H, m*, aromatic). b)To a solution of PSMP-F (1.00 g, 3.22 mmol) in THF (15 mL) at 0° C. wasadded sodium hydride (0.129 g of a 60% mineral oil dispersion). Themixture was stirred for 10 minutes, whereupon a solution ofBoc-homophenylalaninal (0.42 g, 1.59 mmol) in THF (5 mL) was added. Themixture was stirred for 30 minutes. 1M HCl (10 mL) was added. Theproducts,(S)-(E)-1-fluoro-3-tert-butoxycarbonylamino-5-phenyl-1-phenylsulfonyl-1-penteneand(S)-(Z)-1-fluoro-3-tert-butoxycarbonylamino-5-phenyl-1-phenylsulfonyl-1-pentene(Boc-Hph-VSPh-VF)were extracted into ethyl acetate (30 mL), washed with saturated aqueoussodium bicarbonate (10 mL), brine (10 mL), dried over MgSO₄, filtered,and the solvent was removed under reduced pressure. TLC: (30% ethylacetate/hexane) R_(f)=0.29 and 0.39. c) The material from theWadsworth-Emmons coupling was dissolved in ether (2 mL). A solution ofanhydrous 4-toluenesulfonic acid (0.67 g, 3.98 mmol) in ether (2 mL).The mixture was stirred at room temperature overnight. Ether (30 mL) wasadded. The solids were filtered, washed with ether (2×20 mL), and driedin vacuo to give 0.42 g (54%)(S)-(E)-1-fluoro-3-amino-5-phenyl-1-phenylsulfonyl-1-pentene4-toluenesulfonate and(S)-(Z)-1-fluoro-3-amino-5-phenyl-1-phenylsulfonyl-1-pentene4-toluenesulfonate(TsOH.Hph-VSPh-VF) in an approximately 5:3 ratio as evidenced by NMR. ¹HNMR (DMSO-d⁶): 1.82-2.07 (2H, m, CH₂CH₂C₆H₅); 2.28 (3H, s, CH₃C₆H₄SO₃⁻); 2.37-2.73 (2H, m*, CH₂CH₂C₆H₅); 4.02 (0.63H, m*, CHNH (E isomer));4.83 (0.37H, m*, CHNH (Z isomer)); 6.22-6.34 (0.37H, dd, J=6,6 Hz, CH═CF(Z isomer)); 6.43 (0.63H, dd, J=6,18 Hz, CH═CF (trans isomer));7.04-8.04 (14 H, m, aromatic) 8.22 (3H, br s, NH₃ ⁺). d) To a solutionof Mu-PheOH (0.238 g, 0.854 mmol) in THF (5 mL) at −10° C. were added4-methylmorpholine (94 (L, 0.854 mmol) and isobuty) chloroformate (0.111mL, 0.854 mmol). The mixture was stirred for 5 minutes, whereuponTsOH.HphVSPh-VF (0.42 g, 0.854 mmol) was added, followed by4methylmorpholine (94 (L, 0.354 mmol). The mixture was stirred for 45minutes. Ethyl acetate (20 mL) was added. The mixture was washed with 1MHCl (10 mL), saturated aqueous sodium bicarbonate (5 mL). brine (5 mL),dried over MgSO₄, filtered, and the solvent was removed under reducedpressure. Yield=0,32 g (64%).

Example 32 In vitro Activity of Selected Michael Acceptors as CysteineProtease Inhibitors Against the Protozoans of Leishmania donovani,Trypanosoma cruzi, and Trypanosoma brucei

In this table, the in vitro antiprotozoal activities of the indicatedcompounds were measured as percentage inhibition of Leishmania donovaniamastigote infecteda mouse peritoneal macrophages, Trypanosoma cruziamastigote infected macrophages, and Trypanosoma brucei extracellularbloodstream forms. Where indicated, the term “T” indicates the moleculeto have displayed cytotoxicity.

Compound (uM) 90 30 10 3 1 ED50 Mu-Leu-Hph-VSPh L. donovani T 15.3 0 4.6T. cruzi T 11.4 0 0 T. brucei 100 84.7 82.2 11.2 Mu-Phe-HphVSPh L.donovani T 9.4 0 0 T. cruzi T 50.1 10 10 T. brucei 100 100 82.7 90.510.2 2.19 Mu-Phe-ArgVSPh.HBr L. donovani 1.3 0 0 0 T. cruzi 5.8 0 0 0 T.brucei 0 0 0 0 Mu-Phe-Hph-VAMBzl L. donovani 23.3 14.2 0 0 T. cruzi 5.830 0 0 T. brucei 0 0 0 0 Mu-Phe-AlaVSMe L. donovani 6.7 0 0 0 T. cruzi12.2 11.2 0 0 T. brucei 0 0 0 0 HCl.Gly-Phe-VSPh L. donovani 4.02 0 0 T.cruzi 0 0.1 0 0 T. brucei 0 0 0 0 Boc-Asp(O-t-Bu)VSMe L. donovani 12.913.4 0 0 T. cruzi 9.9 0 0 0 T. brucei 0 0 0 0 HCl.PheVSMe L. donovani100 100 100 100 T. cruzi 17.3 0 0 0 T. brucei 0 0 0 0 Mu-Phe-ValVSPh L.donovani T 100 100 100 T. cruzi 74.2 18.9 6.2 5.5 61.88 T. brucei 10072.7 35.4 13.5 11.9 13.85 HCl.(e-Z)LysVSPh L. donovani T T 100 100 T.cruzi 97.8 82.8 10.6 2.7 18.34 T. brucei 100 100 39.3 13 7.6 8Mu-Phe-Ser(OBzl)VSPh L. donovani T 100 100 100 T. cruzi 50.7 15.1 15.75.2 T. brucei 100 1.00 93.3 11.5 5.2 Mu-Phe-(e-Z)LysVSPh L. donovani 0 00 0 T. cruzi 0 0 0 0 T. brucei 100 100 81.3 29.9 19.8 3.64Mu-Phe-HphStyrNO2 L. donovani 20.5 10.8 4 5.3 T. cruzi 12.2 0 0 0 T.brucei 100 100 0 0

Example 33 In vitro Activity of Selected Michael Acceptors as CysteineProtease Inhibitors Against the Malarial Parasites P. falciparum (human)and P. vinckei (mouse)

The following table summarizes the comparative IC₅₀ values for selectedcysteine protease inhibitors against the human malarial parasitePlasmodium falciparum and the mouse malarial parasite Plasmodiumvinckei. The substrate used to measure in vitro protease activity wasZ-Phe-Arg-AMC.

P. falciparum P. vinckei Compound IC₅₀ (human) IC₅₀ (murine)Mu-Phe-AlaVSMe 20 μM Mu-Phe-HphVSMe 1 μM 1 μM Mu-Phe-PheVSMe 2 μMHCl.Ala-PheVSMe >100 μM Mu-Phe-ArgVSPh.HBr 50 nM 50 nM Mu-Phe-HphVSPh 80nM 50 nM Mu-Phe-(e-Z)LysVSPh 100 nM 80 nM Mu-Phe-LysVSPh.HBr 100 nM 60nM Mu-Phe-ValVSPh 1 μM 1 μM Mu-Phe-Ser(OBzl)VSPh 1 μM 500 nMMu-Leu-HphVSPh 3 nM 200 nM Mu-Phe-HphVEOEt 300 nM 300 nMMu-Phe-ArgVEOEt.HBr 300 nM 200 nM Mu-Phe-HphVA 1 μM 2 μMMu-Phe-HphVAMLeu-ProOMe 80 nM 600 nM Mu-Phe-HphVAMBzl 200 nM 2 μMMu-Phe-HphVPEt 3 μM Mu-Phe-HphStyrNO₂ 2 μM 8 μM

Example 34 Treatment of Rheumatoid Arthritis with Peptidyl Vinylsulfones

At day zero, Female Lewis rats (5/group), 5 weeks old, were givenintradermal injection into the base of the tail of Mycobacteriumbutyricum in 0.1 ml of light mineral oil. The animals were providedTeklad (4%) rat chow mixed with (treated groups) or without compound(control group) and water ad libilum. The compounds used wereMu-Phe-HphVSPh, Mu-Leu-HphVSPh, Mu-Tyr-HphVSPh, Mu-Phe-Lys(ε-Z)VSPh,Mu-Phe-LysVSPh-HBr. The compounds were given to three groups at 3, 10,and 30 mg/kg/day, respectively. The following parameters were noted:biweekly weights and biweekly joint evaluations by the following scoringsystem: paw swelling 0=no edema, 1=slight edema of small digital joints,2=edema of the digital joints and foot pad, 3=gross edema of the entirefoot pad below that ankle or elbow, 4=gross edema of the entire foot padincluding the ankle and elbow joint Erythema was scored as: 0=normal,1=pink, 2=red, 3=deep violaceous. The evaluators were blinded as totreatment groups.

After 28 days the animals were killed and the hind paws and knees wereremoved and fixed in 4% paraformaldehyde. The bones were decalcified inFisher Decalcifying Solution (chelating agent, 3 mM HCl 1.35 N) and thenembedded in paraffin (58°, 3×45 min). Joint histopathology scores werecalculated by the following method: two sections of each joint were readfor synovial cell proliferation, cartilage erosion, bone erosion,fibroproliferative pannus, diffuse inflammatory synovitis, and synovialvasculitis: 0=normal, 1=mild, 2=moderate, 3=and severe. The sectionswere examined by two independent investigators blinded as to treatmentgroups, and the mean score then used as the score for that section.

Mu-Phe-HphVSPh showed a dose dependent beneficial effect on all scores.The high dose of Mu-Leu-HphVSPh also showed a reduction in bone andcartilage destruction.

Example 35 Activity of Peptidyl Vinylsulfones Against P. carinii

Mu-Leu-HphVSPh and Mu-Phe-LysVSPh.HBr were evaluated for their effectson the growth of P. carinii in vitro, according to the procedure of M.S. Bartlett et al. [Antimicrotubule Benzimidazoles Inhibit In VitroGrowth of P. carinii, M. S. Bartlett, T. D. Edlind, M. M. Durkin, M. M.Shaw, S. F. Queener, and J. W. Smith, (1992) Antimicrobial Agents andChemotherapy, 36, 779-782; Antimicrobial susceptibility of P. carinii inculture, M. S. Bartlett, R. Eichholtz, and J. W. Smith (1985) Diagn.Microbiol. Infect. Dis.; 3, 381-387. Mu-Leu-HphVSPh andMu-Phe-LysVSPh.HBr (10 mM each) inhibited the growth of P. carinii 64%and 50% respectively.

Example 36 Activity of Peptidyl Vinylsulfones and One Vinyl AmideAgainst T. cruzi

Irradiated J774 cells were infected with T. cruzi and simultaneouslytreated with 20 mM of peptidyl vinyl derivatives for 5 days; thereafterwithout inhibitor. The following compounds were effective in decreasingorder: Mu-Phe-ArgVSPh, Boc-Tyr(I₂)-HphVSPh, Mu-Phe-VaIVSPh,Boc-Tic-HphVSPh, Mu-Phe-Ser(OBzl)VSPh, Mu-Leu-HphVSPh,Mu-Tyr(I₂)-HphVSPh, Mu-Phe-LysVSPh, Mu-Tic-HphVSPh.

Example 37 Effect of Peptidyl Vinylsulfones on Glioma Cell Migration

Two permanent human glioma cell lines (U87MG and U251MGn) and a wellcharacterized low passage primary culture derived from a CB-positiveglioblastoma (HF66) were used to assess the migratory behavior in theMatrigel barrier migration assay in the presence of gradedconcentrations of four compounds.

Using the Matrigel assay, Mu-Leu-HphVSPh, Mu-Tyr-HphVSPh,Mu-Phe-HphVSPh, and Mu-Tyr(I₂)-HphVSPh (all at 10 mM) inhibited U251MGnby 67, 56, 29, & 20% respectively, while the U87MG cells were inhibited53, 75, 63, & 56% respectively. The primary culture HF66 cells wereinhibited only 10% by these compounds at the same concentration.

Example 38 Inhibition of Calpain

The inhibitors were tested against calpain at 1 or 2% as for the otherenzymes. The reaction conditions were 50 mM Tris, pH 7.5, 5 mM CaCl₂,2.5 mM DTT, assayed at 25° C. The substrate was 100 μM Suc-Leu-Tyr-AMC.The results are shown in Table 6.

TABLE 6 Inactivation of Calpain-1, 2% k/K_(I) or k_(obs)/[I]* Inhibitor(M⁻¹sec⁻¹) k_(irr), sec⁻¹ K_(I), μM Boc-D(O-tBu)VSMe  0 — —Mu-DL-Fam-HphVSPh  0 Mu-Tic-HphVSPh  0 — — Boc-Tic-HphVSPh  0 — —Boc-Tyr(I₂)HphVSPh  0 — — Ac-Tyr-Val-Ala-AspVSPh  0 — — Mu-Phe-AlaVSMe<50* — — Mu-Phe-HphVPhe <50* — — Mu-Phe-HphVAMBzl <50* — —Mu-Phe-HphVPhos <50* — — Mu-Phe-KysVSPh <50* — — Mu-Phe-Ser(OBzl)VSPh<50*- — — Mu-Phe-KysVSPh <50* — — Mu-Phe-ArgVSPh <50* — — Mu-Phe-HphVSPh<50* — — Mu-ARg-ArgVSPh <50* — — Mu-Phe-LysVE <50* — — Boc-Ala-PheVSMe 60* — — Phe-VSPh <50* — — Gly-PheVSPh <50* — — Mu-Val-PheVSPh 110* — —Mu-Phe-ValVSPh 180* — — Mu-Phe-PheVSMe 260* — — Ac-Leu-Leu-MetVSPh5,600*  — — Ac-Leu-Leu-NleVSPh 6,900*  — — Mu-Leu-Leu-Tyr-VSPh 7,600*  —— Ac-Leu-Leu- 8,400*  — — Met(O₂)VSPh Mu-Leu-Leu-TyrVSPh 10,800%  — —Z-Leu-Leu-Tyr-VSPh 36,400%  — — Z-leu-Leu-TyrVSPh 24,300   — —

We claim:
 1. A cysteine protease inhibitor of the formula:

wherein EWG is phosphonate, sulfoxide, sulfonamide, sulfinamide,sulfoximine, sulfonate, C(O)OR₁, S(O₂)R₂, C(O)NHCH(Z)C(O)Q, or C(O)R₄;R₁ is (C₅)alkyl, (C₃₋₇)cycloalkyl, (C₃₋₇)cycloalkyl(C₁₋₅)alkyl,optionally substituted aryl, or optionally substituted (C₇₋₁₂)aralkyl,wherein said optional substituents are one or two groups of (C₁₋₅)alkyl,(C₁₋₅)alkoxy, halogen of atomic number 9 to 35, hydroxy, amino, orhalogen substituted (C₁₋₅)alkyl; R₂ is substituted (C₁)alkyl, optionallysubstituted (C₂₋₅)alkyl, (wherein said substituent groups of said(C₁)alkyl and (C₂₋₅)alkyl are (C₁₋₅)alkoxy, halogen of atomic numberfrom 9 to 35, hydroxy, amino, nitro, arylsulfonyl, orhalogen-substituted (C₁₋₅)alkyl), (C₃₋₇)cycloalkyl,(C₃₋₇)cycloalkyl(C₁₋₅)alkyl, (C₃₋₇)cycloalky(C₁₋₅)alkenyl, (C₅₋₁₂)aryl,or (C₇₋₁₂)aralkyl, wherein said (C₅₋₁₂)aryl, or (C₇₋₁₂)aralkyl areoptionally substituted with one or two groups of (C₁₋₅)alkoxy, halogenof atomic number from 9 to 35, hydroxy, amino, nitro, alkyl,arylsulfonyl or halogen-substituted (C₁₋₅)alkyl; Q is hydrogen, ester,NH₂, NH—(C₁₋₅)alkyl NH—(C₃₋₇)cycloalkyl, NH—(C₃₋₇)cycloalky(C₁₋₅)alkyl,NH—C₅₋₁₂)aryl, NH—(C₇₋₁₂)aralkyl, N-di(C₁₋₅)alkyl, N-di(C₃₋₇)cycloalkyl,N-di(C₃₋₇)cycloalkyl(C₁₋₅)alkyl, N-di(C₅₋₁₂)aryl, or N-di(C₇₋₁₂)aralkyl,wherein said aryl and aralkyl groups are optionally substituted with oneor two (C₁₋₅)alkyl, (C₁₋₅)alkoxy, halogen of atomic number 9 to 35,hydroxy, amino, nitro, (C₁₋₅)alkyl, (C₅₋₁₂)arylsulfonyl, orhalogen-substituted (C₁₋₅)alkyl; R₄ is (C₂₋₅)alkyl, (C₃₋₇)cycloalkyl,(C₃₋₇)cycloalky(C₁₋₅)alkyl, (C₃₋₇)cycloalky(C₁₋₅)alkenyl, optionallysubstituted (C₅₋₁₂)aryl, or optionally substituted (C₇₋₁₂)aralkyl,wherein said substituent groups are one or two (C₁₋₅)alkyl,(C₁₋₅)alkoxy, halogen of atomic number 9 to 35, hydroxy, amino, nitro,(C₁₋₅)alkyl, (C₅₋₁₂)arylsulfonyl, halogen-substituted (C₁₋₅)alkyl orperfluoro; R₁₀ is hydrogen, a peptide amino end blocking group, apeptide residue with or without an amino end blocking group, or a singleamino acid with or without an amino end blocking group, wherein saidamino end blocking group is selected from the group consisting ofalkoxy-ω-oxoalkanoyl of 2 to 10 carbon atoms, alkoxycarbonyl of 2 to 10carbon atoms, alkanoyl of 2 to 10 carbon atoms, cycloalkylcarbonyl of 4to 8 carbon atoms, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl,or alkylsulfonyl of 1 to 10 carbon atoms; X, R₁₁, and Z are amino acidside chains in the (R) or (S) configuration; A—B is a peptide linkage orpeptidomimetic linkage; and wherein the second order rate constant forinhibition of a cysteine protease with said inhibitor (k_(irr)/K_(I)) isat least about 1000 M⁻¹sec⁻¹.
 2. The cysteine protease inhibitor ofclaim 1 of the formula:

wherein R₃ is (C₁₋₅)alkyl, (C₃₋₇)cycloalkyl, (C₃₋₇)cycloalky(C₁₋₅)alkyl,(C₃₋₇)cycloalkyl(C₁₋₅)alkenyl, optionally substituted (C₅₋₁₂)aryl, oroptionally substituted (C₇₋₁₂)aralkyl, wherein said substituent groupsare one or two (C₁₋₅)alkyl, (C₁₋₅)alkoxy, halogen of atomic number 9 to35, hydroxy, amino, nitro, (C₁₋₅)alkyl, (C₅₋₁₂)arylsulfonyl,halogen-substituted (C₁₋₅)alkyl, or perfluoro.
 3. The cysteine proteaseinhibitor of claim 1 of the formula:

R₅ is (C₁₋₅)alkyl, (C₃₋₇)cycloalkyl, (C₃₋₇)cycloalkyl(C₁₋₅)alkyl,(C₃₋₇)cycloalkyl(C₁₋₅)alkenyl, optionally substituted (C₅₋₁₂)aryl, oroptionally substituted (C₇₋₁₂)aralkyl, wherein said optionalsubstituents are one to two groups of (C₁₋₅)alkyl, (C₁₋₅)alkoxy, halogenof atomic number of 9 to 35, hydroxy, amino, nitro alkyl, alylsulfonyl,halogen substituted (C₁₋₅)alkyl or perfluoro.
 4. The cysteine proteaseinhibitor of claim 1 of the formula:


5. The cysteine protease inhibitor of claim 1 of the formula:

R₆ and R₇ are independently (C₁₋₅)alkyl, (C₃₋₇)cycloalkyl,(C₃₋₇)cycloalky(C₁₋₅)alkyl, (C₃₋₇)cycloalkyl(C₁₋₅)alkenyl, optionallysubstituted (C₅₋₁₂)aryl, or optionally substituted (C₇₋₁₂)aralkyl,wherein said substituent groups are one or two (C₁₋₅)alkyl,(C₁₋₅)alkoxy, halogen of atomic number 9 to 35, hydroxy, amino, nitro,(C₁₋₅)alkyl, (C₅₋₁₂)arylsulfonyl, halogen-substituted (C₁₋₅)alkyl, orperfluoro.
 6. A cysteine protease inhibitor of the formula:

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, wherein said amino end blockinggroup is selected from the group consisting of alkoxy-ω-oxoalkanoyl of 2to 10 carbon atoms, alkoxycarbonyl of 2 to 10 carbon atoms, alkanoyl of2 to 10 carbon atoms, cycloalkylcarbonyl of 4 to 8 carbon atoms,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl, alkylsulfonyl of 1to 10 carbon atoms; A—B is a peptide linkage or peptidomimetic linkage;R₈ is a five or six membered homocyclic aromatic ring with at least onesubstituted group selected from EWM, MDG, and DG, wherein EWM is ester,sulfone, carboxylate, amide, phosphonate, ketone, nitrile, sulfonate,sulfoxide, sulfonamide, sulfinamide, or sulfoximine; MDG is SO₃H, SO₂R,SOR, SO₂NH₂, SO₂NHR, SO₂N(R)₂, SONH₂, SONHR, SON(R)₂, CN, PO₃H,P(O)(OR)₂, P(O)OR, OH, COOH, COR, COOR, or NRRR⁺, wherein each R isindependently an aryl, alkyl, or aralkyl; and DG is a halogen; andwherein the second order rate constant for inhibition of a cysteineprotease with said inhibitor (k_(irr)/K_(I)) is at least about 1000M⁻¹sec⁻¹.
 7. The cysteine protease inhibitor of claim 6 of the formula:


8. A cysteine protease inhibitor of the formula:

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, wherein said amino end blockinggroup is selected from the group consisting of alkoxy-ω-oxoalkanoyl of 2to 10 carbon atoms, alkoxycarbonyl of 2 to 10 carbon atoms, alkanoyl of2 to 10 carbon atoms, cycloalkylcarbonyl of 4 to 8 carbon atoms,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl, alkylsulfonyl of 1to 10 carbon atoms; X and R₁₁ are amino acid side chains in the (R) or(S) configuration; A—B is a peptide linkage or peptidomimetic linkage;R₈ is a five or six membered homocyclic aromatic ring with at least onesubstituted group selected from EWM, a MDG, and DG, wherein EWM is anester, sulfone, carboxylate, amide, phosphonate, ketone, nitrile, nitrocompound, sulfonate, sulfoxide, sulfonamide, sulfinamide, orsulfoximine; MDG is NO₂, SO₃H, SO₂R, SOR, SO₂NH₂, SO₂NHR, SO₂N(R)₂,SONH₂, SONHR, SON(R)₂, CN, PO₃H, P(O)(OR)₂, P(O)OR, OH, COOH, COR, COOR,or NRRR⁺, wherein each R is independently an aryl, alkyl, or aralkyl;and DG is a halogen; R₉ is methyl or a peptide; and wherein the secondorder rate constant for inhibition of a cysteine protease with saidinhibitor (k_(irr)/K_(I)) is at least about 1000 M⁻¹sec⁻¹.
 9. A cysteineprotease inhibitor of the formula:

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, wherein said amino end blockinggroup is selected from the group consisting of alkoxy-ω-oxoalkanoyl of 2to 10 carbon atoms, alkoxycarbonyl of 2 to 10 carbon atoms, alkanoyl of2 to 10 carbon atoms, cycloalkylcarbonyl of 4 to 8 carbon atoms,carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl, alkylsulfonyl of 1to 10 carbon atoms; X and R₁₁ are amino acid side chains in the (R) or(S) configuration; A—B is a peptide linkage or peptidomimetic linkage;R₈ is a five or six membered heterocyclic aromatic ring with at leastone substituted group selected from EWM, a MDG, and DG, wherein EWM isan ester, sulfone, carboxylate, amide, phosphonate, ketone, nitrile,nitro compound, sulfonate, sulfoxide, sulfonamide, sulfinamide, orsulfoximine; MDG is NO₂, SO₃H, SO₂R, SOR, SO₂NH₂, SO₂NHR, SO₂N(R)₂,SONH₂, SONHR SON(R)₂, CN, PO₃H, P(O)(OR)₂, P(O)OR, OH, COOH, COR, COOR,or NRRR⁺, wherein each R is independently an aryl, alkyl, or aralkyl;and DG is a halogen; R₉ is hydrogen, methyl or a peptide; and whereinthe second order rate constant for inhibition of a cysteine proteasewith said inhibitor (k_(irr)/K_(I)) is at least about 1000 M⁻¹sec⁻¹. 10.A cysteine protease inhibitor of claim 9 of the formula:

wherein D is oxygen, sulfur, nitrogen, phosphorus or arsenic.
 11. Thecysteine protease inhibitor of claim 9 of the formula:

wherein T is nitrogen or phosphorus.
 12. A cysteine protease inhibitorof the formula:

wherein R₁₀ is hydrogen, a peptide amino end blocking group, a peptideresidue with or without an amino end blocking group, a single amino acidwith or without an amino end blocking group wherein said amino endblocking group is selected from the group consisting ofalkoxy-ω-oxoalkanoyl of 2 to 10 carbons, alkoxycarbonyl of 2 to 10carbons, alkanoyl of 2 to 10 carbons, cycloalkylcarbonyl of 4 to 8carbons, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl, oralkylsulfonyl of 1 to 10 carbons; X and R₁₁ are amino acid side chainsin the (R) or (S) configuration; A—B is a peptide linkage orpeptidomimetic linkage; EWG represents an ester, a sulfone, carboxylate,phosphonate, an amide, a ketone, a nitrite, a sulfoxide, a sulfoximine,a five or six membered homocyclic or heterocyclic aromatic ring with atleast one substituted group selected from EWM, MDG, and DG, wherein EWMis an ester, sulfone, carboxylate, amide, phosphonate ketone, nitrite,nitro compound, sulfonate, sulfoxide, sulfonamide, sulfinamide, orsulfoximine; MDG is NO₂, SO₃H, SO₂R, SOR, SO₂NH₂, SO₂NHR, SO₂N(R)₂,SONH₂, SONHR, SON(R)₂, CN, PO₃H, P(O)(OR)₂, P(O)OR, OH, COOH, COR, COOR,or NRRR⁺, wherein each R is independently an aryl, alkyl, or aralkyl;and DG is a halogen; and wherein the second order rate constant forinhibition of a cysteine protease with said inhibitor (k_(irr)/K_(I)) isat least about 1000 M⁻¹sec⁻¹.
 13. A cysteine protease inhibitor of theformula:

wherein R₁₀ is hydrogen, a peptide amino end blocking group, a peptideresidue with or without an amino end blocking group, a single amino acidwith or without an amino end blocking group wherein said amino endblocking group is selected from the group consisting ofalkoxy-ω-oxoalkanoyl of 2 to 10 carbons, alkoxycarbonyl of 2 to 10carbons, alkanoyl of 2 to 10 carbons, cycloalkylcarbonyl of 4 to 8carbons, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl, oralkylsulfonyl of 1 to 10 carbons; X and R₁₁ are amino acid side chainsin the (R) or (S) configuration; A—B is a peptide linkage orpeptidomimetic linkage; EWG represents an ester, a sulfone, carboxylate,phosphonate, an amide, a ketone, a nitrile, a sulfoxide, a sulfoxamine,a five or six membered homocyclic or heterocyclic aromatic ring with atleast one substituted group selected from EWM, MDG, and DG, wherein EWMis an ester, sulfone, carboxylate, amide, phosphonate, ketone, nitrile,nitro compound, sulfonate, sulfoxide, sulfonamide, sulfinamide, orsulfoximine; MDG is NO₂, SO₃H, SO₂R, SOR, SO₂NH₂, SO₂NHR, SO₂N(R)₂,SONH₂, SONHR, SON(R)₂, CN, PO₃H, P(O)(OR)₂, P(O)OR, OH, COOH, COR, COOR,or NRRR⁻, wherein each R is independently an aryl, alkyl, or aralkyl;and DG is a halogen; and wherein the second order rate constant forinhibition of a cysteine protease with said inhibitor (k_(irr)/K_(I)) isat least about 1000 M⁻¹sec⁻¹.
 14. A cysteine protease inhibitor of theformula:

wherein R₁₀ is selected from the group consisting of hydrogen, a peptideresidue of 1-3 amino acids with or without a peptide end blocking group,and a peptide end blocking group.
 15. A method of inhibiting a cysteineprotease comprising irreversibly binding the cysteine protease inhibitorof claims 1, 6, 8, 9, 12, 13 or 14 to said cysteine protease.
 16. Acysteine protease inhibitor comprising a compound of the formula:

wherein R₁₀ is a pepetide and blocking group.
 17. The cysteine proteaseinhibitor of claim 16 wherein said peptide end blocking group isselected from the group consisting of C₍₂₋₁₀₎alkoxy-ω-oxoalkanoyl,C₍₂₋₁₀₎alkoxycarbonyl, C₍₂₋₁₀₎alkanoyl, C₍₄₋₈₎cycloalkylcarbonyl,carbamoyl, dialkylcarbamoyl, benzoyl, C₍₁₋₁₀₎alkylsulfonyl.heterocycloarylcarbonyl of 4 to 10 atoms in the ring, andheterocycloalkylcarbonyl of 4 to 8 atoms in the ring.
 18. The cysteineprotease inhibitor of claim 17, wherein said C₍₂₋₁₀₎alkoxycarbonyl isC₍₄₋₈₎alkoxycarbonyl.
 19. The cysteine protease inhibitor of claim 18,wherein said C₍₄₋₈₎alkoxycarbonyl is tert-butoxycarbonyl.
 20. Thecysteine protease inhibitor of claim 16 wherein said peptide endblocking group is heterocycloalkylcarbonyl.
 21. The cysteine proteaseinhibitor of claim 20 wherein said heterocycloalkylcarbonyl ismorpholinecarbonyl.
 22. The cysteine protease inhibitor of claim 20wherein said heterocycloalkylcarbonyl is cycloalkylaminocarbonyl. 23.The cysteine protease inhibitor of claim 22 wherein saidcycloalkylaminocarbonyl is cycloalkyldiaminocarbonyl.
 24. The cysteineprotease inhibitor of claim 23 wherein said cycloalkyldiaminocarbonyl is(4-methyl-piperazin-1-yl)-methanoyl.
 25. The cysteine protease inhibitoraccording to claim 24 wherein said cysteine protease inhibitor is4-methylpiperazine-1-carboxylic acid[1-((E)-3-benzenesulfonyl-1-phenethyl-allylcarbamoyl)-2-phenyl-ethyl]-amide.26. The cysteine protease inhibitor of claim 16 wherein said peptide endblocking group is benzyloxycarbonyl.
 27. The cysteine protease inhibitorof claim 16, wherein said peptide end blocking group isheterocycloarylcarbonyl.
 28. The cysteine protease inhibitor of claim27, wherein said heterocycloarylcarbonyl is isoqinolinecarbonyl.
 29. Amethod of making a cysteine protease inhibitor of the formula:

wherein EWG is phosphonate, sulfoxide, sulfonamide, sulfinamide,sulfoximine, sulfonate, C(O)OR₁, S(O₂)R₂, C(O)NHCH(Z)C(O)Q, or C(O)R₄;R₁ is (C₅)alkyl, (C₃₋₇)cycloalkyl, (C₃₋₇)cycloalkyl(C₁₋₅)alkyl,optionally substituted aryl, or optionally substituted (C₇₋₁₂)aralkyl,wherein said optional substituents are one or two groups of (C₁₋₅)alkyl,(C₁₋₅)alkoxy, halogen of atomic number 9 to 35, hydroxy, amino, orhalogen substituted (C₁₋₅)alkyl; R₂ is substituted (C₁)alkyl, optionallysubstituted (C₂₋₅)akyl, (wherein said substituent groups of said(C₁)alkyl and (C₂₋₅)alkyl are (C₁₋₅)alkoxy, halogen of atomic numberfrom 9 to 35, hydroxy, amino, nitro, arylsulfonyl, orhalogen-substituted (C₁₋₅)alkyl), (C₃₋₇)cycloalkyl,(C₃₋₇)cycloalkyl(C₁₋₅)alkyl, (C₃₋₇)cycloalky(C₁₋₅)alkenyl, (C₅₋₁₂)aryl,or (C₇₋₁₂)aralkyl, wherein said (C₅₋₁₂)aryl, or (C₇₋₁₂)aralkyl areoptionally substituted with one or two groups of (C₁₋₅)alkoxy, halogenof atomic number from 9 to 35, hydroxy, amino, nitro, alkyl,arylsulfonyl or halogen-substituted (C₁₋₅)alkyl; Q is hydrogen, ester,NH₂, NH—(C₁₋₅)alkyl NH—(C₃₋₇)cycloalkyl, NH—(C₃₋₇)cycloalky(C₁₋₅)alkyl,NH—(C₅₋₁₂)aryl, NH—(C₇₋₁₂)aralkyl, N-di(C₁₋₅)alkyl,N-di(C₃₋₇)cycloalkyl, N-di(C₃₋₇)cycloalkyl(C₁₋₅)alkyl, N-di(C₅₋₁₂)aryl,or N-di(C₇₋₁₂)aralkyl, wherein said aryl and aralkyl groups areoptionally substituted with one or two (C₁₋₅)alkyl, (C₁₋₅)alkoxy,halogen of atomic number 9 to 35, hydroxy, amino, nitro, (C₁₋₅)alkyl,(C₅₋₁₂)arylsulfonyl, or halogen-substituted (C₁₋₅)alkyl; R₄ is(C₂₋₅)alkyl, (C₃₋₇)cycloalkyl, (C₃₋₇)cycloalky(C₁₋₅)alkyl,(C₃₋₇)cycloalkyl(C₁₋₅)alkenyl, optionally substituted (C₅₋₁₂)aryl, oroptionally substituted (C₇₋₁₂)aralkyl, wherein said substituent groupsare one or two (C₁₋₅)alkyl, (C₁₋₅)alkoxy, halogen of atomic number 9 to35, hydroxy, amino, nitro, (C₁₋₅)alkyl, (C₅₋₁₂)arylsulfonyl,halogen-substituted (C₁₋₅)alkyl or perfluoro; R₁₀ is hydrogen, a peptideamino end blocking group, a peptide residue with or without an amino endblocking group, or a single amino acid with or without an amino endblocking group, or a label, wherein said amino end blocking group isselected from the group consisting of alkoxy-ω-oxoalkanoyl of 2 to 10carbon atoms, alkoxycarbonyl of 2 to 10 carbon atoms, alkanoyl of 2 to10 carbon atoms, cycloalkylcarbonyl of 4 to 8 carbon atoms, carbamoyl,alkylcarbamoyl, dialkylcarbamoyl, benzoyl, or alkylsulfonyl of 1 to 10carbon atoms, and wherein said label is selected from the groupconsisting of isotopic labels, immune labels, colored labels andfluorescent labels; X, R₁₁, and Z are amino acid side chains in the (R)or (S) configuration; A—B is a peptide linkage or peptidomimeticlinkage; and wherein the second order rate constant for inhibition of acysteine protease with said inhibitor (k_(irr)/K_(I)) is at least about1000 M⁻¹sec⁻¹, said method comprising: a) coupling a protected α-aminoR₁₁ aldehyde with a Wadsworth-Emmons reagent containing said EWG to forma cysteine protease inhibitor intermediate; b) deprotecting the cysteineprotease inhibitor intermediate; and c) coupling the cysteine proteaseinhibitor intermediate with an N-protected X amino acid.
 30. A method ofmaking a cysteine protease inhibitor of the formula:

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label, wherein said amino endblocking group is selected from the group consisting ofalkoxy-ω-oxoalkanoyl of 2 to 10 carbon atoms, alkoxycarbonyl of 2 to 10carbon atoms, alkanoyl of 2 to 10 carbon atoms, cycloalkylcarbonyl of 4to 8 carbon atoms, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl,alkylsulfonyl of 1 to 10 carbon atoms, and wherein said label isselected from the group consisting of isotopic labels, immune labels,colored labels and fluorescent labels; X and R₁₁ are amino acid sidechains in the (R) or (S) configuration; A—B is a peptide linkage orpeptidomimetic linkage; R₈ is a five or six membered homocyclic aromaticring with at least one substituted group selected from EWM, MDG, and DG,wherein EWM is ester, sulfone, carboxylate, amide, phosphonate, ketone,nitrile, sulfonate, sulfoxide, sulfonamide, sulfinamide, or sulfoximine;MDG is SO₃H, SO₂R, SOR, SO₂NH₂, SO₂NHR,SO₂N(R)₂, SONH₂, SONHR, SON(R)₂,CN, PO₃H, P(O)(OR)₂, P(O)OR, OH, COOH, COR, COOR, or NRRR⁺, wherein eachR is independently an aryl, alkyl, or aralkyl; and DG is a halogen; andwherein the second order rate constant for inhibition of a cysteineprotease with said inhibitor (k_(irr)/K_(I)) is at least about 1000M⁻¹sec⁻¹, said method comprising: a) coupling a protected α-amino R₁₁aldehyde with a Wadsworth-Emmons reagent containing said R₈ group toform a cysteine protease inhibitor intermediate; b) deprotecting thecysteine protease inhibitor intermediate; and c) coupling the cysteineprotease inhibitor intermediate with an N-protected X amino acid.
 31. Amethod of making a cysteine protease inhibitor of the formula:

R₁₀ is hydrogen, a peptide amino end blocking group, a peptide residuewith or without an amino end blocking group, a single amino acid with orwithout an amino end blocking group, or a label, wherein said amino endblocking group is selected from the group consisting ofalkoxy-ω-oxoalkanoyl of 2 to 10 carbon atoms, alkoxycarbonyl of 2 to 10carbon atoms, alkanoyl of 2 to 10 carbon atoms, cycloalkylcarbonyl of 4to 8 carbon atoms, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl,alkylsulfonyl of 1 to 10 carbon atoms, and wherein said label isselected from the group consisting of isotopic labels, immune labels,colored labels and fluorescent labels; X and R₁₁ are amino acid sidechains in the (R) or (S) configuration; A—B is a peptide linkage orpeptidomimetic linkage; R₈ is a five or six membered homocyclic aromaticring with at least one substituted group selected from EWM, a MDG, andDG, wherein EWM is an ester, sulfone, carboxylate, amide, phosphonate,ketone, nitrile, nitro compound, sulfonate, sulfoxide, sulfonamide,sulfinamide, or sulfoximine; MDG is NO₂, SO₃H, SO₂R, SOR, SO₂NH₂,SO₂NHR, SO₂N(R)₂, SONH₂, SONHR, SON(R)₂, CN, PO₃H, P(O)(OR)₂, P(O)OR,OH, COOH, COR, COOR, or NRRR⁺, wherein each R is independently an aryl,alkyl, or aralkyl; and DG is a halogen; R₉ is methyl or a peptide; andwherein the second order rate constant for inhibition of a cysteineprotease with said inhibitor (k_(irr)/K_(I)) is at least about 1000M⁻¹sec⁻¹, said method comprising: a) coupling a protected α-amino R₁₁aldehyde with a Wadsworth-Emmons reagent containing said R₈ and R₉groups to form a cysteine protease inhibitor intermediate; b)deprotecting the cysteine protease inhibitor intermediate; and c)coupling the cysteine protease inhibitor intermediate with anN-protected X amino acid.
 32. A method of making a cysteine proteaseinhibitor of the formula:

wherein R₁₀ is hydrogen, a peptide amino end blocking group, a peptideresidue with or without an amino end blocking group, a single amino acidwith or without an amino end blocking group or a label, wherein saidamino end blocking group is selected from the group consisting ofalkoxy-ω-oxoalkanoyl of 2 to 10 carbons, alkoxycarbonyl of 2 to 10carbons, alkanoyl of 2 to 10 carbons, cycloalkylcarbonyl of 4 to 8carbons, carbamoyl, alkylcarbamoyl, dialkylcarbamoyl, benzoyl, oralkylsulfonyl of 1 to 10 carbons, and wherein said label is selectedfrom the group consisting of isotopic labels, immune labels, coloredlabels and fluorescent labels; X and R₁₁ are amino acid side chains inthe (R) or (S) configuration; A—B is a peptide linkage or peptidomimeticlinkage; EWG represents an ester, a sulfone, carboxylate, phosphonate,an amide, a ketone, a nitrile, a sulfoxide, a sulfoxamine, a five or sixmembered homocyclic or heterocyclic aromatic ring with at least onesubstituted group selected from EWM, MDG, and DG, wherein EWM is anester, sulfone, carboxylate, amide, phosphonate, ketone, nitrile, nitrocompound, sulfonate, sulfoxide, sulfonamide, sulfinamide, orsulfoximine; MDG is NO₂, SO₃H, SO₂R, SOR, SO₂NH₂, SO₂NHR, SO₂N(R)₂H,SONH₂, SONHR, SON(R)₂, CN, PO₃H, P(O)(OR)₂, P(O)OR, OH, COOH, COR, COOR,or NRRR⁺, wherein each R is independently an aryl, alkyl, or aralkyl;and DG is a halogen; and wherein the second order rate constant forinhibition of a cysteine protease with said inhibitor (k_(irr)/K_(I)) isat least about 1000 M⁻¹sec⁻¹, said method comprising: a) coupling aprotected α-amino R₁₁ vinylogous aldehyde with a Wadsworth-Emmonsreagent containing an EWG to form a diene cysteine protease inhibitorintermediate; b) deprotecting the diene cysteine protease inhibitorintermediate; and c) coupling the diene cysteine protease inhibitorintermediate with an N-protected “X” amino acid.